Azapeptides as CD36 binding compounds

ABSTRACT

An azapeptide compound of Formula I:
 
A-(Xaa) a -N(R A )—N(R B )—C(O)-(Xaa′) b -B   I.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of application Ser. No.13/829,797 (U.S. Patent Application Publication No. 2013-0203686 A1),filed Mar. 14, 2013 (issued as U.S. Pat. No. 9,115,171 on Aug. 25,2015), which is a continuation of application Ser. No. 12/452,114(issued as U.S. Pat. No. 8,435,954 on May 7, 2013), filed on Apr. 26,2010, which is a National Entry Application of PCT application No.PCT/CA2008/001162, filed on Jun. 18, 2008 and published in English underPCT Article 21(2), which claims the benefit of U.S. provisionalapplication No. 60/944,712, filed on Jun. 18, 2007. All documents aboveare incorporated herein in their entirety by reference.

FIELD OF INVENTION

The present invention concerns azapeptides as CD36 binding compounds,and methods of making said azapeptides.

BACKGROUND OF THE INVENTION

Age-related macular degeneration (AMD) is one of the leading causes ofirreversible vision loss in the western world accounting for 75% oflegal blindness of the population of age 50 and older in developedcountries (1). The prevalence of AMD which is of 0.05% before 50 yearsold, rises to 11.8% after 80 years of age and is expected to double inthe coming decades because of the projected increase in agingpopulations (2, 3).

The causes of AMD are poorly understood, but it is agreed that theprogressive decline of vision in AMD results from the dysfunction of thecentral retina principally its underlying elements, the retinal pigmentepithelium (RPE), the Bruch membrane (BM), the choriocapillaris anddegeneration of the photoreceptors (4). Other than age, few predisposingfactors have been clearly identified; these include light, cigarettesmoking, possibly hypertension and atherosclerosis (5). In this context,despite their specific characteristics an analogy between deposits foundin AMD and atherosclerosis has been proposed (6).

Early AMD is characterized by focal or diffuse sub-RPE debris in BM(Drusen and basal deposits respectively), changes in RPE pigmentationand by thinning and obliteration of the choriocapillary layer (4). Twoclinical forms of late AMD are identified: the non-exudative formcharacterized by geographic atrophy of RPE and choroid (geographicatrophy, GA) and the exudative form, which also includes choroidalneovascularisation (CNV) (7). Although the non-exudative form isdisabling due to patchy defects in the central visual field, it is thechoroidal neovascularisation of the exudative form that leads toblindness via its leaky vessels that prone to subretinal exsudations andhemorraghes (1) leading to the destruction of macular photoreceptors.The clinical features common for both types of AMD include thedeposition of amorphous white deposits of phospholipids and oxidizedlipoproteins (drusen), and inflammatory mediators that develop betweenthe RPE and the BM as well as the hypo/hyperpigmentation of the RPE.

Although the underlying pathogenesis and its sequence that leads to AMDis not yet defined, the key pathophysiological steps have beensummarized as 1) impaired transport between the RPE and thechoriocapillaris leads to debris accumulation in the interposed BM, 2)deposition of drusen leads to RPE and photoreceptor degeneration, and 3)deregulation of the balance of pro- and anti-angiogenic factors leadingto choroidal involution or neovascularisation (CNV). As CNV is a keyfactor in preserving vision in the aged population, the development oftherapeutic agents that impairs CNV has been considered for thetreatment of AMD (8).

The development of CNV in AMD has been thought to be induced by thehypoxia due to the reduced diffusion of oxygen and nutrients from thechoroid to the retinal pigment epithelium (RPE) following the thickeningof Bruchs membrane resulting from the deposit of lipid and proteinmaterial (9). This hypoxia conjugated with the choroid hypoperfusioninduces a significant upregulation of the expression of VEGFs and VEGFRsin the RPE cells as well as in the endothelial cells of thechoriocapillaris (10, 11) promoting therefore angiogenesis in agerelated macular degeneration.

The treatment strategies in AMD are mainly targeted to inhibit theocular neovascularisation by blocking the expression or the activity ofVEGFs and its receptors.

The blocking the expression of VEGF and its receptor has been approachedby the silencing RNA technology. Silencing VEGF using SiRNA techniquehas been proposed by Acuity Pharmaceuticals in the development of Cand 5(12). Intravitreal injection of Cand 5 was found to inhibit theneovascular growth without systemic toxicity.

The same approach of the siRNA technique to downregulate the expressionof VEGFR-1 following the intravitreal and periocular injections ofSima-027 has been proposed by SIRNA therapeutics (13). It was foundeffective in reducing choroidal and retinal neovascularisation (14).However, the long-term effect of SiRNA approaches remains to bedocumented.

The most frequent antiangiogenesis approach in the treatment of AMDconsists of the inhibition of VEGF binding using specific aptamer,anti-VEGF antibodies or sVEGFRs.

The first development of aptamer (Pegaptanib), a covalent conjugate ofan oligonucleotide and PEG that binds to the extracellular isoforms ofVEGF was initiated for the treatment of neovascular AMD. Although thisinnovative approach appears highly promising, it does not reduce the CNVdevelopment and is unable to improve overall vision (15).

The anti VEGF therapy using the recombinant humanized Fab derived fromthe anti-VEGF murine monoclonal antibody (Ranibizumab) or thefull-length anti-VEGF monoclonal humanised antibody (Bevacizumab) hasbeen reported to be effective in preventing the formation of CNV (16)with a significant decrease in central retinal thickness. Although, thetherapy using antibodies against VEGF which inhibits all VEGF isoformshas a drawback since VEGF is also a survival factor for neuronal cellsand a fundamental requisite for the maintenance of the fenestration ofthe choriocapillaris which is necessary for the physiological functionof the choroid itself, the retinal pigment epithelium and the outerretina. The chronic inhibition of VEGF could lead to the atrophy ofthese tissues.

The development of a fusion protein featuring a higher binding affinityto VEGF which combines extracellular domains of VEGFR-1 and 2 to The Fcportion on IgG1 (VEGF-TRAP) has been shown to inhibit CNV following itssystemic administration. However, the adverse effect of hypertensivecrisis following systemic administration of this ligand preventedfurther exploration in the treatment of AMD (17).

Blocking VEGF activity by interfering with its signalling pathways hasbeen explored. Effectively, VEGF binding to its receptors leads to thephosphorylation of cytoplasmic signalling proteins such as PI3 kinase,MAP kinase and PKC. The selective inhibition of isoforms of PKC by SU5416 (18) (a VEGF inhibitor) or PKC412 (19) reduces CNV development withless angiographic leakage. However, systemic adverse reactions such asnausea and hepatic toxicity have been reported.

The inhibition of the cellular effect of VEGF with the use ofintravitreal steroids has been considered for the treatment ofneovascular AMD and exudative retinal diseases. Triamcinolone acetonidehas been shown to feature angiostatic effect in animal models with CNV(20). The combination of intravitreal steroid treatment withphotodynamic therapy appears to give better vision outcomes (21).However, the major disavantages of such treatment consist of the rise ofintraocular pressure with the progression of cataracts (22). The newgeneration of modified steroids (cortesines) such as Anecortave acetate,which is devoid of glucocorticoid and mineralocorticoid activitiesresponsible for the steroid-associated adverse effects is in evaluationfor the prevention of AMD development (23).

Thus, there is still a need to develop antiangiogenic strategies to stopthe neovascular growth and leakage in the treatment of AMD. Recentreports have shown that the accumulation of oxidized lipoproteins in theRPE cells and Bruchs membrane, which is consistent with the accumulationof cholesterol esters and phospholipid-containing debris in the Bruchsmembrane, is paralleled with that of macrophages in the AMD lesions(24). The macrophages express scavenger receptors and may accumulate forthe uptake of oxidized lipoproteins. Suppressing the macrophageaccumulation by controlling macrophage responses to oxidativelipoproteins and phospholipid oxidation might be complementary for thetreatment of AMD (24).

Among the seven families known of scavenger receptors, CD36 a type Bscavenger receptor has been shown to be involved in multiple functions:(1) cellular energy uptake as a long chain fatty acid (LCFA) receptor(25), (2) clearance of oxidized low density lipoprotein (oxLDL) (26),(3) phagocytosis of retinal outer segments (ROS) for the recycling ofspent photoreceptor disks (27), (4) mediation of the antiangiogeniceffect of thrombospondin-1 (28). Interestingly, CD36 was found expressedin RPE, microvascular endothelial cells and in microglia (29) which aremajor cell types in AMD as well as in macrophages found in CNV membranes(24). Its expression could be upregulated by oxLDL and by otheroxidative and oxidation-prone products including docosahexaenoic acids apredominant fatty acid in retinal tissue particularly the outer segment(30). As the oxidation process increases with age, oxidized lipoproteinsare internalized for subsequent degradation by these cells. A deficiencyin the clearance of these oxidized lipids as observed in the LDL-R nullor ApoE null mice (31, 32) resulted in the accumulation of debris(drusen) in subRPE and BM. The localization of CD36, its scavengingfunction towards oxidized lipids and its modulatory role inangiogenesis, makes this receptor an interesting potential candidate forthe genesis of AMD by way of lipid build up in BM, retinal degeneration,and vascular obliteration resulting ultimately in the development ofneovascularisation.

Growth hormone-releasing peptides (GHRPs) consist of a family of smallsynthetic peptides derived from en kephalins that were developed asgrowth hormone secretagogues (33). These peptides feature high affinitybinding to the ghrelin receptor (GHS-R1a) a G-coupled receptor mainlyexpressed in the hypothalamus and are involved in the stimulation ofgrowth hormone-release (34). Besides their endocrine activity, GHRPsfeature GH-independent cardioprotective activity in improvingpost-ischemic cardiac dysfunction (35) and antiatherosclerotic activity,preventing the development of atherosclerotic plaques in the ApoE nullmice model (36). This beneficial effect appears to be CD36-dependent andmight be due, at least in part, to the reduction of the oxLDL uptake bymacrophages and to the increase of cholesterol and phospholipid effluxfrom macrophages through the activation of transcription factors PPARyand LXRa and the ABC transporters (37). The peripheral activity of GHRPsmight be mediated by their interaction with the scavenger receptor CD36as shown by covalent photolabelling study with a photoactivatablederivative of hexarelin, the hexapeptide prototype of GHRPs (38) whichbinds also with high affinity to the GHS-R1a receptor (34).

SUMMARY OF THE INVENTION

A novel class of azapeptide compounds has been discovered, which areanalogs of growth hormone-releasing peptide-6 (GHRP-6). It has beendemonstrated that the compounds are antiangiogenic and that they inhibitthe development of choroidal neovascularisation in vivo in a CD36dependent manner. The compounds selectively bind to CD36 with loss ofbinding activity at the ghrelin receptor GHS-R1a as shown by bindingstudies. Furthermore, the compounds inhibit in vitro vascular sproutingof aortic endothelium. The compounds also inhibit choroidalneovascularisation in vivo as assessed using the model of laser injuryinduced neovascularisation. Thus, the compounds can be used to treatchoroidal neovascularisation in age-related macular degeneration and inother forms of neovascularisation related diseases.

According to an aspect of the present invention there is provided anazapeptide compound of Formula I:A-(Xaa)_(a)-N(R^(A))—N(R^(B))—C(O)-(Xaa′)_(b)-B   I

wherein

a is an integer from 0 to 5;

b is an integer from 0 to 5;

Xaa and Xaa′ are each any D or L amino acid residue or a D,L amino acidresidue mixture;

A is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₄ alkynyl,

5) C₃-C₇ cycloalkyl,

6) haloalkyl,

7) heteroalkyl,

8) aryl,

9) heteroaryl,

10) heteroalkyl,

11) heterocyclyl,

12) heterobicyclyl,

13) C(O)R³,

14) SO₂R³,

15) C(O)OR³, or

16) C(O)NR⁴R⁵,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

B is

1) OH,

2) OR³, or

3) NR⁴R⁵;

R^(A) and R^(B) are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) C₃-C₇ cycloalkyl,

6) C₅-C₇ cycloalkenyl,

7) haloalkyl,

8) heteroalkyl,

9) aryl,

10) heteroaryl,

11) heterobicyclyl, or

12) heterocyclyl,

wherein the alkyl, alkenyl, alkynyl and the cycloalkyl and cycloalkenylare optionally substituted with one or more R¹ substituents; and whereinthe aryl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents,

or alternatively, R^(A) and R^(B) together with the nitrogen to whicheach is bonded form a heterocyclic or a heterobicyclic ring;

R¹ is

1) halogen,

2) NO₂,

3) CN,

4) haloalkyl,

5) C₃-C₇ cycloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl,

9) heterobicyclyl,

10) OR⁶,

11) S(O)₂R³,

12) NR⁴R⁵,

13) NR⁴S(O)₂R³,

14) COR⁶,

15) C(O)OR⁶,

16) CONR⁴R⁵,

17) S(O)₂NR⁴R⁵,

18) OC(O)R⁶,

19) SC(O)R³,

20) NR⁶C(O)NR⁴R⁵,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵;

wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl areoptionally substituted with one or more R² substituents;

R² is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) aryl,

18) heteroaryl,

19) heterocyclyl,

20) heterobicyclyl,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵,

wherein the aryl, the heteroaryl, the heterocyclyl, and theheterobicyclyl are optionally substituted with one or more R⁷substituents;

R³ is

1) C₁-C₆ alkyl,

2) C₂-C₆ alkenyl,

3) C₂-C₄ alkynyl,

4) C₃-C₇ cycloalkyl,

5) haloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl, or

9) heterobicyclyl,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

R⁴ and R⁵ are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl,

or R⁴ and R⁵ together with the nitrogen to which they are bonded form aheterocyclic ring;

R⁶ is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl;

R⁷ is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) heteroalkyl,

18) NR⁶C(NR⁶)NR⁴R⁵, or

19) C(NR⁶)NR⁴R⁵;

or a salt thereof, or a prodrug thereof;

wherein the following compounds are excluded:

A is H, (Xaa)_(a) is His-D-Trp, R^(A) is H, R^(B) is CH₃, (Xaa′)_(b) isTrp-D-Phe-Lys and B is NH₂;

A is H, (Xaa)_(a) is His-D-Trp-Ala-Trp, R^(A) is H, R^(B) is CH₂Ph,(Xaa′)_(b) is Lys and B is NH₂;

A is H, (Xaa)_(a) is (D/L)-His, R^(A) is H, R^(B) is CH₂-p-C₆H₄OH,(Xaa′)_(b) is Ala-Trp-D-Phe-Lys and B is NH₂;

A is H, (Xaa)_(a) is His-D-Trp-Ala, R^(A) is H, R^(B) is CH₂-p-C₆H₄OH,(Xaa′)_(b) is D-Phe-Lys and B is NH₂; and

A is H, (Xaa)_(a) is His-D-Trp-Ala-D-Phe, R^(A) is H, R^(B) is—(CH₂)₄NH₂, _(b) is 0, and B is NH₂.

In one embodiment, a is an integer from 1 to 5 and b is an integer from1 to 5.

In another aspect of the invention, there is provided a pharmaceuticalcomposition comprising a compound of Formula I, as described above, withone or more of a pharmaceutically acceptable carrier, diluent orexcipient.

In another aspect of the invention there is provided a method ofpreparing the pharmaceutical composition as described above, the methodcomprising: mixing the compound of Formula I, as described above, withthe one or more of the pharmaceutically acceptable carrier, diluent orexcipient.

From another aspect, there is also provided is a method of inhibitingCD36-dependent vascularization in vitro or in vivo, inhibiting choroidalneovascularization in vitro or in vivo, or inhibiting angiogenesis invitro or in vivo, the method comprising: contacting CD36 with a compoundof Formula I, as described above, in an amount sufficient to inhibit thevascularization, the choroidal neovascularization or the angiogenesis.

From another aspect, there is also provided a method of treating maculardegeneration in a subject, or treating atheroscleorosis in a subject,the method comprising: administering to the subject in need thereof atherapeutically effective amount of a compound of Formula I, asdescribed above, to treat the macular degeneration or theatheroscleorosis.

From yet another aspect, there is provided a method of detecting CD36 ina biological sample, the method comprising: a) contacting the biologicalsample with an amount of a compound of Formula I, as described above,wherein the compound is detectably labeled; and b) detecting the labeledcompound bound to the CD36.

From a yet further aspect, there is provided a method of inhibitingangiogenesis in vitro or in vivo, inhibiting CD36-dependentvascularization in vitro or in vivo, inhibiting choroidalneovascularization in vitro or in vivo, treating macular degeneration ina subject, or treating atheroscleorosis in a subject, the methodcomprising: contacting CD36 with a compound of Formula I, in an amountsufficient to inhibit angiogenesis, vascularization or choroidalneovascularization, or administering to the subject in need thereof atherapeutically effective amount of a compound of Formula I, to treatthe macular degeneration or the atheroscleorosis, wherein Formula I isdefined as:A-(Xaa)_(a)-N(R^(A))—N(R^(B))—C(O)-(Xaa′)_(b)-B   I

wherein

a is an integer from 0 to 5;

b is an integer from 0 to 5;

Xaa and Xaa′ are each any D or L amino acid residue or a D,L amino acidresidue mixture;

A is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₄ alkynyl,

5) C₃-C₇ cycloalkyl,

6) haloalkyl,

7) heteroalkyl,

8) aryl,

9) heteroaryl,

10) heteroalkyl,

11) heterocyclyl,

12) heterobicyclyl,

13) C(O)R³,

14) SO₂R³,

15) C(O)OR³, or

16) C(O)NR⁴R⁵,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

B is

1) OH,

2) OR³, or

3) NR⁴R⁵;

R^(A) and R^(B) are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) C₃-C₇ cycloalkyl,

6) C₅-C₇ cycloalkenyl,

7) haloalkyl,

8) heteroalkyl,

9) aryl,

10) heteroaryl,

11) heterobicyclyl, or

12) heterocyclyl,

wherein the alkyl, alkenyl, alkynyl and the cycloalkyl and cycloalkenylare optionally substituted with one or more R¹ substituents; and whereinthe aryl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents,

or alternatively, R^(A) and R^(B) together with the nitrogen to whicheach is bonded form a heterocyclic or a heterobicyclic ring;

R¹ is

1) halogen,

2) NO₂,

3) CN,

4) haloalkyl,

5) C₃-C₇ cycloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl,

9) heterobicyclyl,

10) OR⁶,

11) S(O)₂R³,

12) NR⁴R⁵,

13) NR⁴S(O)₂R³,

14) COR⁶,

15) C(O)OR⁶,

16) CONR⁴R⁵,

17) S(O)₂NR⁴R⁵,

18) OC(O)R⁶,

19) SC(O)R³,

20) NR⁶C(O)NR⁴R⁵,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵;

wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl areoptionally substituted with one or more R² substituents;

R² is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) aryl,

18) heteroaryl,

19) heterocyclyl,

20) heterobicyclyl,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵,

wherein the aryl, the heteroaryl, the heterocyclyl, and theheterobicyclyl are optionally substituted with one or more R⁷substituents;

R³ is

1) C₁-C₆ alkyl,

2) C₂-C₆ alkenyl,

3) C₂-C₄ alkynyl,

4) C₃-C₇ cycloalkyl,

5) haloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl, or

9) heterobicyclyl,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

R⁴ and R⁵ are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl,

or R⁴ and R⁵ together with the nitrogen to which they are bonded form aheterocyclic ring;

R⁶ is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl;

R⁷ is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) heteroalkyl,

18) NR⁶C(NR⁶)NR⁴R⁵, or

19) C(NR⁶)NR⁴R⁵;

or a salt thereof, or a prodrug thereof.

In one embodiment, a is an integer from 1 to 5 and b is an integer from1 to 5;

In another aspect of the present invention, there is provided use of acompound of Formula I, as described immediately above, for treatingmacular degeneration, psoriasis, warts, Kaposi Sarcoma, transplantarteriopathy, obesity, tumor growth, allergic dermatis, scar keloids,pyogenic granulomas, retinopathy of prematurity, diabetic retinopathy,primary pulmonary hypertension, asthma, nasal polyps, inflammatorybowel, periodontal disease, ascites, uterine bleeding, endometriosis,persistent hyperplastic vitreous syndrome, ovarian cysts, carvenoushemangioma, synovitis, osteomyelitis, arthritis, atherosclerosis,hypercholesterolemia and cardiovascular disease associated withatherioscierosis.

From another aspect, there is provided a method of solid phase synthesisof azapeptides, such as the azapeptides defined above, the methodcomprising:

a) providing an amino acid or a peptide chain bound to a solid support;

b) constructing an N-terminal semicarbazone onto an amine bound to thesolid support;

c) introducing side chains;

e) deconstruction of the semicarbazones;

f) cleavage of the azapeptides from the support.

The cleavage step can be performed as described in Boeglin et al (39),or any other suitable method.

The construction of an N-terminal semicarbazone onto an amine bound tothe solid support can comprise incorporating activated aza-glycineresidues into the peptide chain or amino acid by treating aldeyhyde orketone-derived hydrazones with a phosgene equivalent. One example of analdeyhyde or ketone-derived hydrazone is arylhydrazones.

Alternatively, this may also be achieved by activation of theresin-bound peptide or amino acid with a phosgene equivalent andtreatment with a hydrazone, or by treating the same intermediate with ahydrazine to form the respective semicarbazide which would be reactedwith an aldehyde or ketone to give the semicarbazone.

The side chains can be introduced by region-selective alkylation of thesemicarbazones.

Alternatively, side chains may be added by a variety of related methodsincluding a) cross-coupling chemistry of the semicarbazone with aryl andheteroaryl halides or triflates, b) Michael additions of semicarbazoneto unsaturated electrophiles such as alpha,beta-unsaturated esters, c)Mitsunobu alkylations with alcohols, or d) alkylations with epoxides andsimilar strained ring systems such as aziridines. Any other suitablemethods are also included.

Deconstruction of the semicarbazones can include reduction of thesemicarbazone. This can be performed using a method similar to thereduction of carbazates as shown in Boeglin et al (39), as well asnucleophilic attack of the semicarbazone by a method similar to thenucleophilic addition to carbazates (as in Friestad, G, Ji, A. Org.Lett.; (Letter); 2008; 10(11); 2311-2313 and refs therein). Moreover,the semicarbazone may serve as an intermediate for heterocycle synthesisat the N-terminal of the peptide chain by methods such as those used onhydrazones like the synthesis of 1,3,4-Trisubstituted Pyrazoles by Deng,X.; Mani, N. S. Org. Lett.; (Letter); 2008; 10(6); 1307-1310.

An optional step of the method may includes acylation of the aza-aminoacid residue, as described in Boeglin et al (39) and Melendez et al(40).

Another optional step may comprise normal SPPS sequence includingcoupling as described in Blankenship et al (68(b)).

The method may further comprise an additional step of protecting a sidegroup followed by deprotecting the side group before cleavage of theazapeptide from the support.

Advantageously, by providing access to a diverse array of aza-amino acidanalogs from a common aza-glycine precursor, this method may be usefulfor studying structure activity relationships to address questions onthe importance of stereochemisty, side-chain functionality andconformation on peptide activity.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent fromthe following description in which reference is made to the appendeddrawings wherein:

FIG. 1a illustrates sprouting aortic endothelium (Von Willebrand factorpositive) expressing CD36 on the upper row compared to vascularsprouting of aortic rings from C57BL/6 and CD36 null mice in presence ofDBG 178 at 10⁻⁷M in the lower row for azapeptide compound DBG178 todemonstrate disruption of vascular sprouting of aortic endothelium;

FIG. 1b is a histogram of microvascular sprouts areas (mean±s.e.m) fromaortic explants of C57BL/6 and CD36 null mice exposed to concentrationsfrom 10⁻⁸ to 10⁻⁶M p<0.01 of DBG 178 compared with control (Ctl);

FIG. 2a illustrates choroidal vessels detected using FITC-conjugateddextran infusion following laser-induced posterior retinal injury ineyes from 10-week-old male C57BL/6 mice and CD36 deficient mice of thesame background with or without intravitreal treatment with azapeptidecompound DBG178;

FIG. 2b is a histogram of the surfaces of neovascular vessels obtainedfrom C57BL/6 (CD36_(+/+)) and CD36 null (CD36_(−/−)) mice with (DBG) orwithout (Ctl) treatment with azapeptide compound DBG178;

FIG. 3 illustrates a general scheme for the synthesis of aza-peptidesaccording to another aspect of the present invention;

FIG. 4 illustrates a general scheme for the activation and coupling ofintermediate 1i to the N-terminus of the peptide bound resin to formsemicarbazone 2ai and 2bi, the synthesis being performed on NovaPEG RinkAmide Resin™;

FIG. 5 illustrates a solution-phase synthesis of benzophenonesemicarbazone aza-Gly-Gly dipeptide 5i and regio-selectivemono-alkylation to yield benzophenone semicarbazone aza-(allyl)Gly-Glydipeptide 6i;

FIG. 6 illustrates a general scheme for the alkylation reaction of FIG.3;

FIG. 7 illustrates a general scheme for the deprotection reaction ofFIG. 3; and

FIG. 8 illustrates an example of the azapeptides synthesized by themethod of FIG. 3.

DETAILED DESCRIPTION

Definitions

Unless otherwise specified, the following definitions apply:

The singular forms “a”, “an” and “the” include corresponding pluralreferences unless the context clearly dictates otherwise.

As used herein, the term “comprising” is intended to mean that the listof elements following the word “comprising” are required or mandatorybut that other elements are optional and may or may not be present.

As used herein, the term “consisting of” is intended to mean includingand limited to whatever follows the phrase “consisting of”. Thus thephrase “consisting of” indicates that the listed elements are requiredor mandatory and that no other elements may be present.

As used herein, the term “amino acid” is intended to mean at least anyof the following □-amino acids:

Amino acid Abbreviation Alanine Ala Arginine Arg Aspartic acid AspAsparagine Asn Cysteine Cys Glutamic acid Glu Glutamine Gln Glycine GlyIsoleucine Ile Histidine His Leucine Leu Lysine Lys Methionine MetPhenylalanine Phe Proline Pro Serine Ser Threonine Thr Tryptophan TrpTyrosine Tyr Valine Val

The above list is not exclusive and it should be understood that otheramino acids not listed above are included in the definition of aminoacid, such as hydroxyproline, citruline, ornithine etc.

The natural amino acids, with the exception of glycine, contain a chiralcarbon atom. Unless otherwise stated, the compounds of Formula Icontaining amino acids can be of either the L- or D-configuration, orcan be mixtures of D- and L-isomers, including racemic mixtures.Additional non-natural amino acid residues which are contemplatedinclude, but are not limited to, □-alkyl, □□□-dialkyl, □-aryl and□-heteroarylglycine analogs, aryl and heteroarylalanine analogs,□□□-dialkylcysteine analogs, □□□-dialkylserine analogs, branched leucineanalogs, ornithine, cirtuline, sarcosine, allylglycine, aminobutyricacid, amino-iso-butyric acid, cyclohexylalanine, cyclohexylglycine (alsonamed: 2-amino-2-cyclohexylacetic acid), norvaline, pipecolic acid,tert-butylglycine, and the like. Also included are □amino acids such asbeta-alanine, beta-homophenylalanine as well as longer chain amino acidssuch as gamma-aminobutyric acid.

As used herein, the term “residue” when referring to □-amino acids isintended to mean a radical derived from the corresponding □-amino acidby eliminating the hydroxyl of the carboxy group and one hydrogen of the□-amino group. For example, the terms Gln, Ala, Gly, Ile, Arg, Asp, Phe,Ser, Leu, Cys, Asn, and Tyr represent the residues of glutamine,alanine, glycine, isoleucine, arginine, aspartic acid, phenylalanine,serine, leucine, cysteine, asparagine, and tyrosine, respectively.

As used herein, the term “alkyl” is intended to include both branchedand straight chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms, for example, C₁-C₆ as in C₁-C₆-alkylis defined as including groups having 1, 2, 3, 4, 5 or 6 carbons in alinear or branched arrangement. Examples of alkyl as defined aboveinclude, but are not limited to, methyl, ethyl, n-propyl, i-propyl,n-butyl, t-butyl, i-butyl, pentyl and c-hexyl.

As used herein, the term, “alkenyl” is intended to mean unsaturatedstraight or branched chain hydrocarbon groups having the specifiednumber of carbon atoms therein, and in which at least two of the carbonatoms are bonded to each other by a double bond, and having either E orZ regeochemistry and combinations thereof. For example, C₂-C₆ as inC₂-C₆ alkenyl is defined as including groups having 1, 2, 3, 4, 5, or 6carbons in a linear or branched arrangement, at least two of the carbonatoms being bonded together by a double bond. Examples of C₂-C₆ alkenylinclude ethenyl (vinyl), 1-propenyl, 2-propenyl, 1-butenyl and the like.

As used herein, the term “alkynyl” is intended to mean unsaturated,straight chain hydrocarbon groups having the specified number of carbonatoms therein and in which at least two carbon atoms are bonded togetherby a triple bond. For example C₂-C₄ as in C₂-C₄ alkynyl is defined asincluding groups having 2, 3, or 4 carbon atoms in a chain, at least twoof the carbon atoms being bonded together by a triple bond.

As used herein, the term “cycloalkyl” is intended to mean a monocyclicsaturated aliphatic hydrocarbon group having the specified number ofcarbon atoms therein, for example, C₃-C₇ as in C₃-C₇cycloalkyl isdefined as including groups having 3, 4, 5, 6 or 7 carbons in amonocyclic arrangement. Examples of C₃-C₇ cycloalkyl as defined aboveinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and cycloheptyl.

As used herein, the term “cycloalkenyl” is intended to mean unsaturatedstraight or branched chain hydrocarbon groups having the specifiednumber of carbon atoms therein in a monocyclic arrangement, and in whichat least two of the carbon atoms are bonded to each other by a doublebond. For example, C₂-C₈ as in C₂-C₈ cycloalkenyl is defined as having2, 3, 4, 5, 6, 7 or 8 carbons in a monocyclic arrangement. Examples ofcycloalkenyls as defined above include, but are not limited tocyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl,cyclooctenyl, and cyclooctadienyl.

As used herein, the term “halo” or “halogen” is intended to meanfluorine, chlorine, bromine and iodine.

As used herein, the term “haloalkyl” is intended to mean an alkyl, asdefined above, in which each hydrogen atom may be successively replacedby a different halogen atom. Examples of haloalkyls include, but are notlimited to, CH₂F, CHF₂ and CF₃.

As used herein, the term “heteroalkyl” is intended to mean a saturatedlinear or branched-chain monovalent hydrocarbon radical of one to sixcarbon atoms, wherein at least one of the carbon atoms is replaced witha heteroatom selected from N, O, or S, and wherein the radical may be acarbon radical or heteroatom radical (i.e., the heteroatom may appear inthe middle or at the end of the radical). The heteroalkyl radical may beoptionally substituted independently with one or more substituentsdescribed herein.

As used herein, the term “aryl”, either alone or in combination withanother radical, means a carbocyclic aromatic monocyclic groupcontaining 6 carbon atoms, which may be further fused to a second 5- or6-membered carbocyclic group which may be aromatic, saturated orunsaturated. Aryl includes, but is not limited to, phenyl, indanyl,1-naphthyl, 2-naphthyl and tetrahydronaphthyl. The fused aryls may beconnected to another group either at a suitable position on the

cycloalkyl ring or the aromatic ring. For example:

Arrowed lines drawn from the ring system indicate that the bond may beattached to any of the suitable ring atoms.

As used herein, the term “heteroaryl” is intended to mean a monocyclicor bicyclic ring system of up to ten atoms, wherein at least one ring isaromatic, and contains from 1 to 4 hetero atoms selected from the groupconsisting of O, N, and S. The heteroaryl substituent may be attachedeither via a ring carbon atom or one of the heteroatoms. Examples ofheteroaryl groups include, but are not limited to thienyl,benzimidazolyl, benzo[b]thienyl, furyl, benzofuranyl, pyranyl,isobenzofuranyl, chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl,imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl,4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, napthyridinyl,quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, isothiazolyl,isochromanyl, chromanyl, isoxazolyl, furazanyl, indolinyl, isoindolinyl,thiazolo[4,5-b]-pyridine, hydroxybenzotriazolyl, benzotriazoyl,triazoyl, and fluorescein derivatives such as:

and rhodamine, dansyl and other fluorescent tags known to those skilledin the art.

As used herein, the term “heterocycle”, “heterocyclic” or “heterocyclyl”is intended to mean a non-aromatic ring system containing heteroatomsselected from the group consisting of O, N and S. Examples of aromaticheterocycles are described as heteroaromatic above. Examples ofnon-aromatic heterocycles include, but are not limited to azepinyl,azetidyl, aziridinyl, pyrrolidinyl, tetrahydrofuranyl, piperidyl,pyrrolinyl, piperazinyl, imidazolidinyl, morpholinyl, imidazolinyl,diazepinyl, pyrazolidinyl, pyrazolinyl, and biotinyl derivatives.

As used herein, the term “heterobicycle” either alone or in combinationwith another radical, is intended to mean a heterocycle as defined abovefused to another cycle, be it a heterocycle, an aryl or any other cycledefined herein. Examples of such heterobicycles include, but are notlimited to, pyrrolizidinyl, indolizidinyl, quinolizidinyl, coumarin,benzo[d][1,3]dioxole, 2,3-dihydrobenzo[b][1,4]dioxine and3,4-dihydro-2H-benzo[b][1,4]dioepine.

As used herein, the term “detectable label” is intended to mean a groupthat may be linked to an azapeptide compound of the present invention toproduce a probe or to a CD36 binding domain, such that when the probe isassociated with the CD36 binding domain, the label allows either director indirect recognition of the probe so that it may be detected,measured and quantified.

As used herein, the term “affinity tag” is intended to mean a ligand orgroup, which is linked to either an azapeptide compound of the presentinvention or to a CD36 binding domain to allow another compound to beextracted from a solution to which the ligand or group is attached.

As used herein, the term “probe” is intended to mean an azapeptidecompound of Formula I which is labeled with either a detectable label oran affinity tag, and which is capable of binding, either covalently ornon-covalently, to a CD36 binding domain. When, for example, the probeis non-covalently bound, it may be displaced by a test compound. When,for example, the probe is bound covalently, it may be used to formcross-linked adducts, which may be quantified and inhibited by a testcompound.

As used herein, the term “optionally substituted with one or moresubstituents” or its equivalent term “optionally substituted with atleast one substituent” is intended to mean that the subsequentlydescribed event of circumstances may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. The definition is intended to meanfrom zero to five substituents.

If the substituents themselves are incompatible with the syntheticmethods of the present invention, the substituent may be protected witha suitable protecting group (PG) that is stable to the reactionconditions used in these methods. The protecting group may be removed ata suitable point in the reaction sequence of the method to provide adesired intermediate or target compound. Suitable protecting groups andthe methods for protecting and de-protecting different substituentsusing such suitable protecting groups are well known to those skilled inthe art; examples of which may be found in T. Greene and P. Wuts,Protecting Groups in Chemical Synthesis (3^(rd) ed.), John Wiley & Sons,NY (1999), which is incorporated herein by reference in its entirety.Examples of protecting groups used throughout include, but are notlimited to Alloc, Fmoc, Bn, Boc, CBz and COCF₃. In some instances, asubstituent may be specifically selected to be reactive under thereaction conditions used in the methods of this invention. Under thesecircumstances, the reaction conditions convert the selected substituentinto another substituent that is either useful in an intermediatecompound in the methods of this invention or is a desired substituent ina target compound.

As used herein, the term “subject” is intended to mean humans andnon-human mammals such as primates, cats, dogs, swine, cattle, sheep,goats, horses, rabbits, rats, mice and the like.

As used herein, the term “prodrug” is intended to mean a compound thatmay be converted under physiological conditions or by solvolysis to abiologically active compound of the present invention. Thus, the term“prodrug” refers to a precursor of a compound of the invention that ispharmaceutically acceptable. A prodrug may be inactive or displaylimited activity when administered to a subject in need thereof, but isconverted in vivo to an active compound of the present invention.Typically, prodrugs are transformed in vivo to yield the compound of theinvention, for example, by hydrolysis in blood or other organs byenzymatic processing. The prod rug compound often offers advantages ofsolubility, tissue compatibility or delayed release in the subject (see,Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier,Amsterdam). The definition of prodrug includes any covalently bondedcarriers, which release the active compound of the invention in vivowhen such prodrug is administered to a subject. Prodrugs of a compoundof the present invention may be prepared by modifying functional groupspresent in the compound of the invention in such a way that themodifications are cleaved, either in routine manipulation or in vivo, toa parent compound of the invention.

As used herein, the term “pharmaceutically acceptable carrier, diluentor excipient” is intended to mean, without limitation, any adjuvant,carrier, excipient, glidant, sweetening agent, diluent, preservative,dye/colorant, aerosol spray, flavor enhancer, surfactant, wetting agent,dispersing agent, suspending agent, stabilizer, isotonic agent, solvent,emulsifier, or encapsulating agent, such as a liposome, cyclodextrins,encapsulating polymeric delivery systems or polyethylene glycol matrix,which is acceptable for use in the subject, preferably humans.

As used herein, the term “pharmaceutically acceptable salt” is intendedto mean both acid and base addition salts.

As used herein, the term “pharmaceutically acceptable acid additionsalt” is intended to mean those salts which retain the biologicaleffectiveness and properties of the free bases, which are notbiologically or otherwise undesirable, and which are formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid and the like, and organic acids suchas acetic acid, trifluoroacetic acid, propionic acid, glycolic acid,pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like.

As used herein, the term “pharmaceutically acceptable base additionsalt” is intended to mean those salts which retain the biologicaleffectiveness and properties of the free acids, which are notbiologically or otherwise undesirable. These salts are prepared fromaddition of an inorganic base or an organic base to the free acid. Saltsderived from inorganic bases include, but are not limited to, thesodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc,copper, manganese, aluminum salts and the like. Salts derived fromorganic bases include, but are not limited to, salts of primary,secondary, and tertiary amines, substituted amines including naturallyoccurring substituted amines, cyclic amines and basic ion exchangeresins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins and the like.

As used herein, the term “therapeutically effective amount” is intendedto mean an amount of an azapeptide compound of Formula I which, whenadministered to a subject is sufficient to effect treatment for adisease-state in which modulation of CD36 activity, such as inhibitionor activation, is desired (The activation of CD36 by the endogenousligand TSP-1 induces the apoptosis of endothelial cells). The amount ofthe compound of Formula I will vary depending on the compound, thecondition and its severity, and the age of the subject to be treated,but can be determined routinely by one of ordinary skill in the arthaving regard to his own knowledge and to this disclosure.

As used herein, the term “treating” or “treatment” is intended to meantreatment of a disease-state in which inhibition of CD36 activity isdesired, as disclosed herein, in a subject, and includes, for example:(i) preventing a disease or condition, in which inhibition of CD36activity is desired, from occurring in a subject, in particular, whensuch mammal is predisposed to the disease or condition but has not yetbeen diagnosed as having it; (ii) inhibiting a disease or conditionassociated with CD36 activity, i.e., arresting its development; or (iii)relieving a disease or condition associated with CD36 activity, i.e.,causing regression of the condition.

As used herein, the term “IC₅₀” is intended to mean an amount,concentration or dosage of a particular compound of the presentinvention that achieves a 50% inhibition of a maximal response measuredunder the same experimental conditions but in the absence of thecompound.

As used herein, the term “EC₅₀” is intended to mean an amount,concentration or dosage of a particular compound of the presentinvention that achieves a 50% inhibition of cell survival measured underthe same experimental conditions but in the absence of the compound.

The compounds of the present invention, or their pharmaceuticallyacceptable salts may contain one or more asymmetric centers, chiral axesand chiral planes and may thus give rise to enantiomers, diastereomers,and other stereoisomeric forms and may be defined in terms of absolutestereochemistry, such as (R)- or (S)- or, as (D)- or (L)-for aminoacids. The present invention is intended to include all such possibleisomers, as well as, their epimeric, racemic and optically pure forms.Optically active (+) and (−), (R)- and (S)-, or (D)- and (L)-isomers maybe prepared using chiral synthons or chiral reagents, or resolved usingconventional techniques, such as reverse phase HPLC. The racemicmixtures may be prepared and thereafter separated into individualoptical isomers or these optical isomers may be prepared by chiralsynthesis. The enantiomers may be resolved by methods known to thoseskilled in the art, for example by formation of diastereoisomeric saltswhich may then be separated by crystallization, gas-liquid or liquidchromatography, selective reaction of one enantiomer with an enantiomerspecific reagent. It will also be appreciated by those skilled in theart that where the desired enantiomer is converted into another chemicalentity by a separation technique, an additional step is then required toform the desired enantiomeric form. Alternatively specific enantiomersmay be synthesized by asymmetric synthesis using enantiomerically purereagents, substrates, catalysts, or solvents or by converting oneenantiomer to another by asymmetric transformation.

Certain compounds of the present invention may exist in Zwitterionicform and the present invention includes Zwitterionic forms of thesecompounds and mixtures thereof.

-   I: Azapeptide Compounds

This invention relates to a novel class of azapeptide compounds ofFormula I which are derivatives of GHRP-6 and which bind specifically tothe scavenger receptor CD36. The selective replacement of amino acids ofGHRP-6 by aza-aminoacid residue in promoting the aromatic interactionsat the ends of the peptide chain improves the binding selectivitytowards the scavenger receptor CD36 and to decrease binding affinity forthe ghrelin receptor GSH-R1a.

One subset of compounds of Formula I comprise compounds in which Xaa isXaa₁, Xaa₂, Xaa₃ or Xaa₄, wherein Xaa₁, Xaa₂, Xaa₃ and Xaa₄ are each a Dor L amino acid residue, and in which Xaa′ is Xaa′₁, Xaa′₂, Xaa′₃ orXaa′₄, wherein Xaa₁, Xaa₂, Xaa₃ and Xaa₄ are each a D or L amino acidresidue.

One subset of azapeptide compounds of Formula I comprise azapeptidecompounds of the following Formula I.1;A-Xaa₁-N(R^(A))—N(R^(B))—C(O)-Xaa′₁-Xaa′₂-Xaa′₃-Xaa′₄-B   I.1

wherein

Xaa₁, Xaa′₁, Xaa′₂, Xaa′₃, and Xaa′₄ are each any D or L amino acidresidue or a D,L amino acid residue mixture;

A is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₄ alkynyl,

5) C₃-C₇ cycloalkyl,

6) haloalkyl,

7) heteroalkyl,

8) aryl,

9) heteroaryl,

10) heteroalkyl,

11) heterocyclyl,

12) heterobicyclyl,

13) C(O)R³,

14) SO₂R³,

15) C(O)OR³, or

16) C(O)NR⁴R⁵,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

B is

1) OH,

2) OR³, or

3) NR⁴R⁵;

R^(A) and R^(B) are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) C₃-C₇ cycloalkyl,

6) C₆-C₇ cycloalkenyl,

7) haloalkyl,

8) heteroalkyl,

9) aryl,

10) heteroaryl,

11) heterobicyclyl, or

12) heterocyclyl,

wherein the alkyl, alkenyl, alkynyl and the cycloalkyl and cycloalkenylare optionally substituted with one or more R¹ substituents; and whereinthe aryl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents,

or alternatively, R^(A) and R^(B) together with the nitrogen to whicheach is bonded form a heterocyclic or a heterobicyclic ring;

R¹ is

1) halogen,

2) NO₂,

3) CN,

4) haloalkyl,

5) C₃-C₇ cycloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl,

9) heterobicyclyl,

10) OR⁶,

11) S(O)₂R³,

12) NR⁴R⁵,

13) NR⁴S(O)₂R³,

14) COR⁶,

15) C(O)OR⁶,

16) CONR⁴R⁵,

17) S(O)₂NR⁴R⁵,

18) OC(O)R⁶,

19) SC(O)R³,

20) NR⁶C(O)NR⁴R⁵,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵;

wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl areoptionally substituted with one or more R² substituents;

R² is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) aryl,

18) heteroaryl,

19) heterocyclyl,

20) heterobicyclyl,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵,

wherein the aryl, the heteroaryl, the heterocyclyl, and theheterobicyclyl are optionally substituted with one or more R⁷substituents;

R³ is

1) C₁-C₆ alkyl,

2) C₂-C₆ alkenyl,

3) C₂-C₄ alkynyl,

4) C₃-C₇ cycloalkyl,

5) haloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl, or

9) heterobicyclyl,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

R⁴ and R⁵ are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl,

or R⁴ and R⁵ together with the nitrogen to which they are bonded form aheterocyclic ring;

R⁶ is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl;

R⁷ is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) heteroalkyl,

18) NR⁶C(NR⁶)NR⁴R⁵, or

19) C(NR⁶)NR⁴R⁵;

or a salt thereof, or a prodrug thereof.

One subset of azapeptide compounds of Formula I comprise azapeptidecompounds of the following Formula I.2:A-Xaa₁-Xaa₂-N(R^(A))—N(R^(B))—C(O)-Xaa′₂-Xaa′₃-Xaa′₄-B   I.2

wherein

Xaa₁, Xaa₂, Xaa′₂, Xaa′₃, and Xaa′₄ are each any D or L amino acidresidue or a D,L amino acid residue mixture;

A is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₄ alkynyl,

5) C₃-C₇ cycloalkyl,

6) haloalkyl,

7) heteroalkyl,

8) aryl,

9) heteroaryl,

10) heteroalkyl,

11) heterocyclyl,

12) heterobicyclyl,

13) C(O)R³,

14) SO₂R³,

15) C(O)OR³, or

16) C(O)NR⁴R⁵,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

B is

1) OH,

2) OR³, or

3) NR⁴R⁵;

R^(A) and R^(B) are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) C₃-C₇ cycloalkyl,

6) C₅-C₇ cycloalkenyl,

7) haloalkyl,

8) heteroalkyl,

9) aryl,

10) heteroaryl,

11) heterobicyclyl, or

12) heterocyclyl,

wherein the alkyl, alkenyl, alkynyl and the cycloalkyl and cycloalkenylare optionally substituted with one or more R¹ substituents; and whereinthe aryl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents,

or alternatively, R^(A) and R^(B) together with the nitrogen to whicheach is bonded form a heterocyclic or a heterobicyclic ring;

R¹ is

1) halogen,

2) NO₂,

3) CN,

4) haloalkyl,

5) C₃-C₇ cycloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl,

9) heterobicyclyl,

10) OR⁶,

11) S(O)₂R³,

12) NR⁴R⁵,

13) NR⁴S(O)₂R³,

14) COR⁶,

15) C(O)OR⁶,

16) CONR⁴R⁵,

17) S(O)₂NR⁴R⁵,

18) OC(O)R⁶,

19) SC(O)R³,

20) NR⁶C(O)NR⁴R⁵,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵;

wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl areoptionally substituted with one or more R² substituents;

R² is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) aryl,

18) heteroaryl,

19) heterocyclyl,

20) heterobicyclyl,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵,

wherein the aryl, the heteroaryl, the heterocyclyl, and theheterobicyclyl are optionally substituted with one or more R⁷substituents;

R³ is

1) C₁-C₆ alkyl,

2) C₂-C₆ alkenyl,

3) C₂-C₄ alkynyl,

4) C₃-C₇ cycloalkyl,

5) haloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl, or

9) heterobicyclyl,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

R⁴ and R⁵ are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl,

or R⁴ and R⁵ together with the nitrogen to which they are bonded form aheterocyclic ring;

R⁶ is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl;

R⁷ is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) heteroalkyl,

18) NR⁶C(NR⁶)NR⁴R⁵, or

19) C(NR⁶)NR⁴R⁵;

or a salt thereof, or a prodrug thereof.

One subset of azapeptide compounds of Formula I comprise azapeptidecompounds of the following Formula I.3;A-Xaa₁-Xaa₂-Xaa₃-N(R^(A))—N(R^(B))—C(O)-Xaa′₃-Xaa′₄-B   I.3

wherein

Xaa₁, Xaa₂, Xaa₃, Xaa′₃, and Xaa′₄ are each any D or L amino acidresidue or a D,L amino acid residue mixture;

A is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₄ alkynyl,

5) C₃-C₇ cycloalkyl,

6) haloalkyl,

7) heteroalkyl,

8) aryl,

9) heteroaryl,

10) heteroalkyl,

11) heterocyclyl,

12) heterobicyclyl,

13) C(O)R³,

14) SO₂R³,

15) C(O)OR³, or

16) C(O)NR⁴R⁵,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

B is

1) OH,

2) OR³, or

3) NR⁴R⁵;

R^(A) and R^(B) are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) C₃-C₇ cycloalkyl,

6) C₅-C₇ cycloalkenyl,

7) haloalkyl,

8) heteroalkyl,

9) aryl,

10) heteroaryl,

11) heterobicyclyl, or

12) heterocyclyl,

wherein the alkyl, alkenyl, alkynyl and the cycloalkyl and cycloalkenylare optionally substituted with one or more R¹ substituents; and whereinthe aryl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents,

or alternatively, R^(A) and R^(B) together with the nitrogen to whicheach is bonded form a heterocyclic or a heterobicyclic ring;

R¹ is

1) halogen,

2) NO₂,

3) CN,

4) haloalkyl,

5) C₃-C₇ cycloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl,

9) heterobicyclyl,

10) OR⁶,

11) S(O)₂R³,

12) NR⁴R⁵,

13) NR⁴S(O)₂R³,

14) COR⁶,

15) C(O)OR⁶,

16) CONR⁴R⁵,

17) S(O)₂NR⁴R⁵,

18) OC(O)R⁶,

19) SC(O)R³,

20) NR⁶C(O)NR⁴R⁵,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵;

wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl areoptionally substituted with one or more R² substituents;

R² is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) aryl,

18) heteroaryl,

19) heterocyclyl,

20) heterobicyclyl,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵,

wherein the aryl, the heteroaryl, the heterocyclyl, and theheterobicyclyl are optionally substituted with one or more R⁷substituents;

R³ is

1) C₁-C₆ alkyl,

2) C₂-C₆ alkenyl,

3) C₂-C₄ alkynyl,

4) C₃-C₇ cycloalkyl,

5) haloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl, or

9) heterobicyclyl,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

R⁴ and R⁵ are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl,

or R⁴ and R⁵ together with the nitrogen to which they are bonded form aheterocyclic ring;

R⁶ is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl;

R⁷ is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) heteroalkyl,

18) NR⁶C(NR⁶)NR⁴R⁵, or

19) C(NR⁶)NR⁴R⁵;

or a salt thereof, or a prodrug thereof.

One subset of azapeptide compounds of Formula I comprise azapeptidecompounds of the following Formula I.4:A-Xaa₁-Xaa₂-Xaa₃-Xaa₄-N(R^(A))—N(R^(B))—C(O)-Xaa′₄-B   I.4

wherein

Xaa₁, Xaa₂, Xaa₃, Xaa₄ and Xaa′₄ are each any D or L amino acid residueor a D,L amino acid residue mixture;

A is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₄ alkynyl,

5) C₃-C₇ cycloalkyl,

6) haloalkyl,

7) heteroalkyl,

8) aryl,

9) heteroaryl,

10) heteroalkyl,

11) heterocyclyl,

12) heterobicyclyl,

13) C(O)R³,

14) SO₂R³,

15) C(O)OR³, or

16) C(O)NR⁴R⁵,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

B is

1) OH,

2) OR³, or

3) NR⁴R⁵;

R^(A) and R^(B) are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) C₃-C₇ cycloalkyl,

6) C₅-C₇ cycloalkenyl,

7) haloalkyl,

8) heteroalkyl,

9) aryl,

10) heteroaryl,

11) heterobicyclyl, or

12) heterocyclyl,

wherein the alkyl, alkenyl, alkynyl and the cycloalkyl and cycloalkenylare optionally substituted with one or more R¹ substituents; and whereinthe aryl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents,

or alternatively, R^(A) and R^(B) together with the nitrogen to whicheach is bonded form a heterocyclic or a heterobicyclic ring;

R¹ is

1) halogen,

2) NO₂,

3) CN,

4) haloalkyl,

5) C₃-C₇ cycloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl,

9) heterobicyclyl,

10) OR⁶,

11) S(O)₂R³,

12) NR⁴R⁵,

13) NR⁴S(O)₂R³,

14) COR⁶,

15) C(O)OR⁶,

16) CONR⁴R⁵,

17) S(O)₂NR⁴R⁵,

18) OC(O)R⁶,

19) SC(O)R³,

20) NR⁶C(O)NR⁴R⁵,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵;

wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl areoptionally substituted with one or more R² substituents;

R² is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) aryl,

18) heteroaryl,

19) heterocyclyl,

20) heterobicyclyl,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵,

wherein the aryl, the heteroaryl, the heterocyclyl, and theheterobicyclyl are optionally substituted with one or more R⁷substituents;

R³ is

1) C₁-C₆ alkyl,

2) C₂-C₆ alkenyl,

3) C₂-C₄ alkynyl,

4) C₃-C₇ cycloalkyl,

5) haloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl, or

9) heterobicyclyl,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

R⁴ and R⁵ are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl,

or R⁴ and R⁵ together with the nitrogen to which they are bonded form aheterocyclic ring;

R⁶ is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl;

R⁷ is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) heteroalkyl,

18) NR⁶C(NR⁶)NR⁴R⁵, or

19) C(NR⁶)NR⁴R⁵;

or a salt thereof, or a prodrug thereof.

One subset of azapeptide compounds of Formula I comprise azapeptidecompounds of the following Formula I.5:A-Xaa₁-N(R^(A))—N(R^(B))—C(O)-Xaa′₂-Xaa′₃-Xaa′₄-B   I.5

wherein

Xaa₁, Xaa′₂, Xaa′₃, and Xaa′₄ are each any D or L amino acid residue ora D,L amino acid residue mixture;

A is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₄ alkynyl,

5) C₃-C₇ cycloalkyl,

6) haloalkyl,

7) heteroalkyl,

8) aryl,

9) heteroaryl,

10) heteroalkyl,

11) heterocyclyl,

12) heterobicyclyl,

13) C(O)R³,

14) SO₂R³,

15) C(O)OR³, or

16) C(O)NR⁴R⁵,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

B is

1) OH,

2) OR³, or

3) NR⁴R⁵;

R^(A) and R^(B) are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) C₃-C₇ cycloalkyl,

6) C₅-C₇ cycloalkenyl,

7) haloalkyl,

8) heteroalkyl,

9) aryl,

10) heteroaryl,

11) heterobicyclyl, or

12) heterocyclyl,

wherein the alkyl, alkenyl, alkynyl and the cycloalkyl and cycloalkenylare optionally substituted with one or more R¹ substituents; and whereinthe aryl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents,

or alternatively, R^(A) and R^(B) together with the nitrogen to whicheach is bonded form a heterocyclic or a heterobicyclic ring;

R¹ is

1) halogen,

2) NO₂,

3) CN,

4) haloalkyl,

5) C₃-C₇ cycloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl,

9) heterobicyclyl,

10) OR⁶,

11) S(O)₂R³,

12) NR⁴R⁵,

13) NR⁴S(O)₂R³,

14) COR⁶,

15) C(O)OR⁶,

16) CONR⁴R⁵,

17) S(O)₂NR⁴R⁵,

18) OC(O)R⁶,

19) SC(O)R³,

20) NR⁶C(O)NR⁴R⁵,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵;

wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl areoptionally substituted with one or more R² substituents;

R² is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) aryl,

18) heteroaryl,

19) heterocyclyl,

20) heterobicyclyl,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵,

wherein the aryl, the heteroaryl, the heterocyclyl, and theheterobicyclyl are optionally substituted with one or more R⁷substituents;

R³ is

1) C₁-C₆ alkyl,

2) C₂-C₆ alkenyl,

3) C₂-C₄ alkynyl,

4) C₃-C₇ cycloalkyl,

5) haloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl, or

9) heterobicyclyl,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

R⁴ and R⁵ are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl,

or R⁴ and R⁵ together with the nitrogen to which they are bonded form aheterocyclic ring;

R⁶ is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl;

R⁷ is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) heteroalkyl,

18) NR⁶C(NR⁶)NR⁴R⁵, or

19) C(NR⁶)NR⁴R⁵;

or a salt thereof, or a prodrug thereof,

One subset of azapeptide compounds of Formula I comprise azapeptidecompounds of the following Formula I.6:A-Xaa₁-Xaa₂-N(R^(A))—N(R^(B))—C(O)-Xaa′₃-Xaa′₄-B  I.6

wherein

Xaa₁, Xaa₂, Xaa′₃, and Xaa′₄ are each any D or L amino acid residue or aD,L amino acid residue mixture;

A is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₄ alkynyl,

5) C₃-C₇ cycloalkyl,

6) haloalkyl,

7) heteroalkyl,

8) aryl,

9) heteroaryl,

10) heteroalkyl,

11) heterocyclyl,

12) heterobicyclyl,

13) C(O)R³,

14) SO₂R³,

15) C(O)OR³, or

16) C(O)NR⁴R⁵,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

B is

1) OH,

2) OR³, or

3) NR⁴R⁵;

R^(A) and R^(B) are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) C₃-C₇ cycloalkyl,

6) C₅-C₇ cycloalkenyl,

7) haloalkyl,

8) heteroalkyl,

9) aryl,

10) heteroaryl,

11) heterobicyclyl, or

12) heterocyclyl,

wherein the alkyl, alkenyl, alkynyl and the cycloalkyl and cycloalkenylare optionally substituted with one or more R¹ substituents; and whereinthe aryl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents,

or alternatively, R^(A) and R^(B) together with the nitrogen to whicheach is bonded form a heterocyclic or a heterobicyclic ring;

R¹ is

1) halogen,

2) NO₂,

3) CN,

4) haloalkyl,

5) C₃-C₇ cycloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl,

9) heterobicyclyl,

10) OR⁶,

11) S(O)₂R³,

12) NR⁴R⁵,

13) NR⁴S(O)₂R³,

14) COR⁶,

15) C(O)OR⁶,

16) CONR⁴R⁵,

17) S(O)₂NR⁴R⁵,

18) OC(O)R⁶,

19) SC(O)R³,

20) NR⁶C(O)NR⁴R⁵,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵;

wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl areoptionally substituted with one or more R² substituents;

R² is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) aryl,

18) heteroaryl,

19) heterocyclyl,

20) heterobicyclyl,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵,

wherein the aryl, the heteroaryl, the heterocyclyl, and theheterobicyclyl are optionally substituted with one or more R⁷substituents;

R³ is

1) C₁-C₆ alkyl,

2) C₂-C₆ alkenyl,

3) C₂-C₄ alkynyl,

4) C₃-C₇ cycloalkyl,

5) haloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl, or

9) heterobicyclyl,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

R⁴ and R⁵ are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl,

or R⁴ and R⁵ together with the nitrogen to which they are bonded form aheterocyclic ring;

R⁶ is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl;

R⁷ is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) heteroalkyl,

18) NR⁶C(NR⁶)NR⁴R⁵, or

19) C(NR⁶)NR⁴R⁵;

or a salt thereof, or a prodrug thereof.

One subset of azapeptide compounds of Formula I comprise azapeptidecompounds of the following Formula I.7:A-Xaa₁-Xaa₂-Xaa₃-N(R^(A))—N(R^(B))—C(O)-Xaa′₄-B  I.7

wherein

Xaa₁, Xaa₂, Xaa₃, and Xaa′₄ are each any D or L amino acid residue or aD,L amino acid residue mixture;

A is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₄ alkynyl,

5) C₃-C₇ cycloalkyl,

6) haloalkyl,

7) heteroalkyl,

8) aryl,

9) heteroaryl,

10) heteroalkyl,

11) heterocyclyl,

12) heterobicyclyl,

13) C(O)R³,

14) SO₂R³,

15) C(O)OR³, or

16) C(O)NR⁴R⁵,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

B is

1) OH,

2) OR³, or

3) NR⁴R⁵;

R^(A) and R^(B) are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) C₃-C₇ cycloalkyl,

6) C₅-C₇ cycloalkenyl,

7) haloalkyl,

8) heteroalkyl,

9) aryl,

10) heteroaryl,

11) heterobicyclyl, or

12) heterocyclyl,

wherein the alkyl, alkenyl, alkynyl and the cycloalkyl and cycloalkenylare optionally substituted with one or more R¹ substituents; and whereinthe aryl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents,

or alternatively, R^(A) and R^(B) together with the nitrogen to whicheach is bonded form a heterocyclic or a heterobicyclic ring;

R¹ is

1) halogen,

2) NO₂,

3) CN,

4) haloalkyl,

5) C₃-C₇ cycloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl,

9) heterobicyclyl,

10) OR⁶,

11) S(O)₂R³,

12) NR⁴R⁵,

13) NR⁴S(O)₂R³,

14) COR⁶,

15) C(O)OR⁶,

16) CONR⁴R⁵,

17) S(O)₂NR⁴R⁵,

18) OC(O)R⁶,

19) SC(O)R³,

20) NR⁶C(O)NR⁴R⁵,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵;

wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl areoptionally substituted with one or more R² substituents;

R² is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) aryl,

18) heteroaryl,

19) heterocyclyl,

20) heterobicyclyl,

21) heteroalkyl,

22) NR⁶C(NR⁶)NR⁴R⁵, or

23) C(NR⁶)NR⁴R⁵,

wherein the aryl, the heteroaryl, the heterocyclyl, and theheterobicyclyl are optionally substituted with one or more substituents;

R³ is

1) C₁-C₆ alkyl,

2) C₂-C₆ alkenyl,

3) C₂-C₄ alkynyl,

4) C₃-C₇ cycloalkyl,

5) haloalkyl,

6) aryl,

7) heteroaryl,

8) heterocyclyl, or

9) heterobicyclyl,

wherein the alkyl, the alkenyl, the alkynyl and the cycloalkyl areoptionally substituted with one or more R¹ substituents; and wherein thearyl, the heteroaryl, the heterocyclyl and the heterobicyclyl areoptionally substituted with one or more R² substituents;

R⁴ and R⁵ are independently chosen from

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl,

or R⁴ and R⁵ together with the nitrogen to which they are bonded form aheterocyclic ring;

R⁶ is

1) H,

2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

4) C₂-C₆ alkynyl,

5) aryl,

6) heteroaryl, or

7) heterocyclyl;

R⁷ is

1) halogen,

2) NO₂,

3) CN,

4) C₁-C₆ alkyl,

5) C₂-C₆ alkenyl,

6) C₂-C₄ alkynyl,

7) C₃-C₇ cycloalkyl,

8) haloalkyl,

9) OR⁶,

10) NR⁴R⁵,

11) SR⁶,

12) COR⁶,

13) C(O)OR⁶,

14) S(O)₂R³,

15) CONR⁴R⁵,

16) S(O)₂NR⁴R⁵,

17) heteroalkyl,

18) NR⁶C(NR⁶)NR⁴R⁵, or

19) C(NR⁶)NR⁴R⁵;

or a salt thereof, or a prodrug thereof,

In one subset of the aforesaid azapeptide compounds of Formula I.1, I.2,I.3 and I.4, A is H such that Xaa₁ is any D or L amino acid residue, ora mixture thereof, with a free N-terminal NH₂ group.

In another subset of the aforesaid azapeptide compounds of Formula I.1,I.2, I.3 and I.4, B is NH₂, such that Xaa′₄ is any D or L amino acidresidue, or a mixture thereof, with a C-terminal CONH₂ group.

The following compounds are excluded:

A is H, (Xaa)_(a) is His-D-Trp, R^(A) is H, R^(B) is CH₃, (Xaa′)_(b) isTrp-D-Phe-Lys and B is NH₂;

A is H, (Xaa)_(a) is His-D-Trp-Ala-Trp, R^(A) is H, R^(B) is CH₂Ph,(Xaa′)_(b) is Lys and B is NH₂;

A is H, (Xaa)_(a) is (D/L)-His, R^(A) is H, R^(B) is CH₂-p-C₆H₄OH,(Xaa′)_(b) is Ala-Trp-D-Phe-Lys and B is NH₂;

A is H, (Xaa)_(a) is His-D-Trp-Ala, R^(A) is H, R^(B) is CH₂-p-C₆H₄OH,(Xaa′)_(b) is D-Phe-Lys and B is NH₂; and

A is H, (Xaa)_(a) is His-D-Trp-Ala-D-Phe, R^(A) is H, R^(B) is—(CH₂)₄NH₂, _(b) is 0, and B is NH₂.

The antiangiogenic property of the azapeptide compounds of the presentinvention have been demonstrated in vitro and in vivo. One compound, DBG178 (see Table 1), has been shown to disrupt significantly the vascularsprouting in the matrigel-embedded aortic rings exposed toconcentrations of this compound ranging from 10⁻⁷ to 10⁻⁶M. Thisinhibitory effect of DBG178, which was not detected in the sprouting ofaortic endothelium from CD36 gene knockout mice, appears to be dependentof CD36 expression, DBG178 has also been shown to inhibit the choroidalneovascularisation in vivo using the laser injury inducedneovascularisation model. In this model, which can be applied to rodentsand primates, a laser beam is used to disrupt the RPE and Bruchsmembrane that separates the choroidal vasculature from the subretinalspace. The following local inflammatory reaction in the deep retina andchoroid leads to a localized subretinal neovascularisation in a similarmanner as that observed in age-related macular degeneration. This localneovascularisation can be quantified on flatmounts ofRPE/choroid/sclera. The intravitreal injection of DBG178 at effectiveconcentrations of 10⁻⁷ and 10⁻⁶ M within the eye 3 and 7 days after thelaser injury showed that DBG 178 significantly inhibited the neovascularresponse by more than 50%. In contrast, DBG178 had no effect on theneovascular response in the eye from CD 36 null mice. Taking togetherthese results, one could conclude that the DBG 178 effect is mediatedthrough the scavenger receptor CD36. DBG178 also efficiently reduces theexaggerated neovascularisation induced by the laser injury in the eye ofC57/BL6 mice. In contrast, the much less neovascularisation observed inthe eye of CD36 null mice following laser injury is not affected by theintravitreal injection of DBG178.

-   II: Utilities

The azapeptide compounds of the present invention are useful asantiangiogenic compounds by modulating CD36 activity, and as such thecompounds, compositions and methods of the present invention includeapplications to the cells or subjects afflicted with or having apredisposition towards developing a particular disease state, for whichinhibition of CD36 activity is desired. For example, the compounds,compositions and methods of the present invention can be used to treatdiseases involving neovascularisation. Besides the subchoroidalneovascularisation in AMD, intravitreal neovascularisation is alsoobserved in diabetic retinopathy, in the retinopathy of prematurity aswell as in retinal vein occlusion. The compounds, compositions andmethods of the present invention may inhibit the neovascularisation insuch pathological conditions.

The GHRP derivative, EP 80317 was shown to serve as a ligand of CD36,and to display anti-inflammatory activity, reducing the expression ofproxydative enzymes in the arterial wall (gp 91 phos and p40 phos of theNADPH oxidase, and i-Nos synthase) as well as causing the reduction ofMCP-1 and VCAM, the chemokines involved in the inflammatory process. Theoverall effect observed was the reduction of atherosclerosis development(36). The modulation of CD36 activity by its ligand (such as GHRP orazapeptide) might result in the reduction of the inflammatory processencountered in inflammation related diseases such as inflammatory boweland arthritis as well as atherosclerosis. As angiogenesis is a majorcontributor in the development of tumors, ligands of CD36 elicitantiangiogenic activity associated with the reduction of tumor growth.The natural ligand TSP-1 is known to bind CD36 at the site of itsextracellular domain (93-110). Azapeptides and GHRPs, that may bind atsites distinct from that used by TSP-1, elicit antiangiogenic effects.The modulation of the activity of CD36 by its ligand may also find itsapplication in the reduction of inflammatory processes encountered ininflammation related diseases such as inflammatory bowel, synovitis,osteomyelitis.

Thus, the compounds, compositions and methods of the present inventioncan be used to treat macular degeneration (whether age-related or not),psoriasis, warts, Kaposi Sarcoma, transplant arteriopathy, obesity,tumor growth, allergic dermatis, scar keloids, pyogenic granulomas,retinopathy of prematurity, diabetic retinopathy, primary pulmonaryhypertension, asthma, nasal polyps, inflammatory bowel and periodontaldisease, ascites, uterine bleeding, endometriosis, persistenthyperplastic vitreous syndrome, ovarian cysts, carvenous hemangioma,synovitis, osteomyelitis, arthritis, and atherosclerosis, which arediseases characterized by excessive or abnormal neovascularization. Inaddition, the compounds, compositions and methods can be used in theprevention of hypercholesterolemia and cardiovascular disease associatedwith atheriosclerosis. The compounds, compositions and methods of thepresent invention can also be used to treat dry macular degeneration(Houssier et al, PLOS Medicine, February 2008, Volume 5, Issue 2).

The treatment involves administration to a subject in need thereof acompound of the present invention or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition comprising a pharmaceuticalcarrier and a therapeutically effective amount of a compound of thepresent invention, or a pharmaceutically acceptable salt thereof.

The compounds of the present invention, or their pharmaceuticallyacceptable salts or their prodrugs, may be administered in pure form orin an appropriate pharmaceutical composition, and can be carried out viaany of the accepted modes of Galenic pharmaceutical practice.

The pharmaceutical compositions of the present invention can be preparedby mixing a compound of the present invention with at least one of anappropriate pharmaceutically acceptable carrier, diluent or excipient,and may be formulated into preparations in solid, semi-solid, liquid orgaseous forms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. Typical routes of administering such pharmaceuticalcompositions include, without limitation, oral, topical, transdermal,inhalation, parenteral (subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques),sublingual, ocular, rectal, vaginal, intranasal and intravitreal.Pharmaceutical compositions of the present invention are formulated soas to allow the active ingredients contained therein to be bioavailableupon administration of the composition to a subject. Compositions thatwill be administered to a subject or patient take the form of one ormore dosage units, where for example, a tablet may be a single dosageunit, and a container of a compound of the present invention in aerosolform may hold a plurality of dosage units. Actual methods of preparingsuch dosage forms are known, or will be apparent, to those skilled inthis art; for example, see Remington's Pharmaceutical Sciences, 18thEd., (Mack Publishing Company, Easton, Pa., 1990). The composition to beadministered will, in any event, contain a therapeutically effectiveamount of a compound of the present invention, or a pharmaceuticallyacceptable salt thereof, for treatment of a disease-state as describedabove.

A pharmaceutical composition of the present invention may be in the formof a solid or liquid. In one aspect, the carrier(s) are particulate, sothat the compositions are, for example, in tablet or powder form. Thecarrier(s) may be liquid, with the compositions being, for example, anoral syrup, injectable liquid or an aerosol, which is useful in, forexample inhalatory administration.

For oral administration, the pharmaceutical composition is preferably ineither solid or liquid form, where semi-solid, semi-liquid, suspensionand gel forms are included within the forms considered herein as eithersolid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like form. Such a solidcomposition will typically contain one or more inert diluents or ediblecarriers. In addition, one or more of the following may be present:binders such as carboxymethylcellulose, ethyl cellulose,microcrystalline cellulose, gum tragacanth or gelatin; excipients suchas starch, lactose or dextrins, disintegrating agents such as alginicacid, sodium alginate, Primogel, corn starch and the like; lubricantssuch as magnesium stearate or Sterotex; glidants such as colloidalsilicon dioxide; sweetening agents such as sucrose or saccharin; aflavoring agent such as peppermint, methyl salicylate or orangeflavoring; and a coloring agent.

When the pharmaceutical composition is in the form of a capsule, e.g., agelatin capsule, it may contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol or oil such assoybean or vegetable oil.

The pharmaceutical composition may be in the form of a liquid, e.g., anelixir, syrup, solution, emulsion or suspension. The liquid may be fororal administration or for delivery by injection, as two examples. Whenintended for oral administration, preferred composition contain, inaddition to the present compounds, one or more of a sweetening agent,preservatives, dye/colorant and flavor enhancer. In a compositionintended to be administered by injection, one or more of a surfactant,preservative, wetting agent, dispersing agent, suspending agent, buffer,stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions of the present invention, whetherthey be solutions, suspensions or other like form, may include one ormore of the following adjuvants: sterile diluents such as water forinjection, saline solution, preferably physiological saline, Ringer'ssolution, isotonic sodium chloride, fixed oils such as synthetic mono ordiglycerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediamine tetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. Solubilization agents may includecyclodextrins such as hydroxypropyl-beta-cyclodextrin. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic. An injectable pharmaceuticalcomposition is preferably sterile.

A liquid pharmaceutical composition of the present invention used foreither parenteral or oral administration should contain an amount of acompound of the present invention such that a suitable dosage will beobtained. Typically, this amount is at least 0.01% of a compound of thepresent invention in the composition. When intended for oraladministration, this amount may be varied to be between 0.1 and about70% of the weight of the composition. For parenteral usage, compositionsand preparations according to the present invention are prepared so thata parenteral dosage unit contains between 0.01 to 1% by weight of thecompound of the present invention.

The pharmaceutical composition of the present invention may be used fortopical administration, in which case the carrier may suitably comprisea solution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of the compound of the present invention from about 0.1 toabout 10% w/v (weight per unit volume). Azapeptide administration by thenasal route is also applicable.

Furthermore, a solution of the azapeptide compound in sterile isotonicsolution may be used for intravitreal injection. A carrier may also beused to transport the azapeptide compound through the sclera, using forexample a biopolymer as the carrier.

The pharmaceutical composition of the present invention may includevarious materials, which modify the physical form of a solid or liquiddosage unit. For example, the composition may include materials thatform a coating shell around the active ingredients. The materials thatform the coating shell are typically inert, and may be selected from,for example, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredients may be encased in a gelatincapsule.

The pharmaceutical composition of the present invention in solid orliquid form may include an agent that binds to the compound of thepresent invention and thereby assists in the delivery of the compound.Suitable agents that may act in this capacity include, but are notlimited to, a monoclonal or polyclonal antibody, a protein or aliposome.

The pharmaceutical compositions of the present invention may consist ofdosage units that can be administered as an aerosol. The term aerosol isused to denote a variety of systems ranging from those of colloidalnature to systems consisting of pressurized packages. Delivery may be bya liquefied or compressed gas or by a suitable pump system thatdispenses the active ingredients. Aerosols of compounds of the presentinvention may be delivered in single phase, bi-phasic, or tri-phasicsystems in order to deliver the active ingredient(s). Delivery of theaerosol includes the necessary container, activators, valves,subcontainers, and the like, which together may form a kit. One skilledin the art, without undue experimentation may determine preferredaerosols.

The pharmaceutical compositions of the present invention may be in aform suitable for ocular administration such as periocular injections orother methods for dispensing compounds and compositions in the eye. Thecompounds or compositions of the present invention could be administeredin solutions, suspensions, in particulate drug delivery systems or inocular implants, for example.

In particulate drug delivery systems, the compounds or compositions ofthe present invention can be incorporated in nanoparticles,microparticles, nanospheres, microspheres, nanocapsules andmicrocapsules (see for example, T Moritera et al. Invest. Opthalmol.Visual Sci 33-3125-3130 (1992); and J L Bourges et al. Invest Opthalmol.Visual science 44-3562-69 (2003)).

Intraocular drug delivery implants could also be used to deliver thecompounds or compositions of the present invention, which may comprisenon-biodegradable solid implants consisting of polyvinylalcohol-ethylenevinyl acetate or polysulfone capillary fiber, for example. Theseimplants could be implanted in the posterior segment of the eyeintravitreally or intrasclerally (see for example, Okabe K et al. InvestOpthalmol. Vis. Sci 44-2702-79 (2003)). Biodegradable solid implants ofpolylactic acid, poly glycolic acid, poly lactic-co-glycolic acid,polycaprolactones or polyanhydrides could also be used (see for example,Yasukawa T et al. Adv Drug Deliv Rev. 57: 2033-46 (2005)).

The compounds and compositions of the present invention could also beincorporated in viscous and injectable poly ortho esters derivatives forintraocular administration (see for example, Einmahl S et al. J. BiomedMater Res. 50: 566-73 (2000); Einmahl S et al. Invest Opthalmol Vis Sci.43: 1533-9 (2002)).

Liposomes, which belong to the family of microparticulate systems, canalso be used as ocular delivery systems or as a microparticulate carriersystem for the compounds and compositions of the present invention (seefor example, Ebrahim S. et al, Surv Opthalmol. 50: 167-82 (2005); andBejjani R^(A) et al. J Fr Opthalmol. 26:981-5 (2003)).

The pharmaceutical compositions of the present invention may be preparedby methodology well known in the pharmaceutical art. For example, apharmaceutical composition intended for administration by injection canbe prepared by admixing a compound of the present invention withsterile, distilled water so as to form a solution. A surfactant may beadded to facilitate the formation of a homogeneous solution orsuspension. Surfactants are compounds that non-covalently interact withthe compound of the present invention so as to facilitate dissolution orhomogeneous suspension of the compound in the aqueous delivery system.

The compounds of the present invention, or their pharmaceuticallyacceptable salts, are administered in a therapeutically effectiveamount, which will vary depending upon a variety of factors includingthe activity of the specific compound employed; the metabolic stabilityand length of action of the compound; the age, body weight, generalhealth, sex, and diet of the patient; the mode and time ofadministration; the rate of excretion; the drug combination; theseverity of the particular disorder or condition; and the subjectundergoing therapy.

-   III: Screening Assays

The compounds of the present invention may also be used in a method toscreen for other compounds that bind to a CD36 binding domain. Generallyspeaking, to use the compounds of the invention in a method ofidentifying compounds that bind to a CD36 binding domain, the CD36 isbound to a support, and a compound of the invention is added to theassay. Alternatively, the compound of the invention may be bound to thesupport and the CD36 is added.

There are a number of ways in which to determine the binding of acompound of the present invention to the CD36 binding domain. In oneway, the compound of the invention, for example, may be fluorescently orradioactively labeled and binding determined directly. For example, thismay be done by attaching the CD36 to a solid support, adding adetectably labeled compound of the invention, washing off excessreagent, and determining whether the amount of the detectable label isthat present on the solid support. Numerous blocking and washing stepsmay be used, which are known to those skilled in the art.

In another example, a gene reporter assay for the screening of CD36ligands is contemplated. The activation of CD36 induces thephosphorylation of src kinases (Fynn. Lynn kinases), and therefore agene luciferase reporter assay would be particularly well suited toscreen CD36 ligands.

The compounds of the invention may also be used as competitors to screenfor additional drug candidates or test compounds. As used herein, theterms “drug candidate” or “test compounds” are used interchangeably anddescribe any molecule, for example, protein, oligopeptide, small organicmolecule, polysaccharide, polynucleotide, and the like, to be tested forbioactivity. The compounds may be capable of directly or indirectlyaltering the CD36 biological activity.

Drug candidates can include various chemical classes, although typicallythey are small organic molecules having a molecular weight of more than100 and less than about 2,500 Daltons. Candidate agents typicallyinclude functional groups necessary for structural interaction withproteins, for example, hydrogen bonding and lipophilic binding, andtypically include at least an amine, carbonyl, hydroxyl, ether, orcarboxyl group. The drug candidates often include cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more functional groups.

Drug candidates can be obtained from any number of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means.

Competitive screening assays may be done by combining a CD36 bindingdomain and a probe to form a probe: CD36 binding domain complex in afirst sample followed by adding a test compound from a second sample.The binding of the test is determined, and a change, or difference inbinding between the two samples indicates the presence of a testcompound capable of binding to the CD36 binding domain and potentiallymodulating the CD36's activity.

In one case, the binding of the test compound is determined through theuse of competitive binding assays. In this embodiment, the probe islabeled with an affinity label such as biotin. Under certaincircumstances, there may be competitive binding between the testcompound and the probe, with the probe displacing the candidate agent.

In one case, the test compound may be labeled. Either the test compound,or a compound of the present invention, or both, is added first to theCD36 binding domain for a time sufficient to allow binding to form acomplex.

Formation of the probe: CD36 binding domain complex typically requireincubations of between 4° C. and 40° C. for between 10 minutes to about1 hour to allow for high-throughput screening. Any excess of reagentsare generally removed or washed away. The test compound is then added,and the presence or absence of the labeled component is followed, toindicate binding to the CD36 binding domain.

In one case, the probe is added first, followed by the test compound.Displacement of the probe is an indication the test compound is bindingto the CD36 binding domain and thus is capable of binding to, andpotentially modulating, the activity of the CD36. Either component canbe labeled. For example, the presence of probe in the wash solutionindicates displacement by the test compound. Alternatively, if the testcompound is labeled, the presence of the probe on the support indicatesdisplacement.

In one case, the test compound may be added first, with incubation andwashing, followed by the probe. The absence of binding by the probe mayindicate the test compound is bound to the CD36 binding domain with ahigher affinity. Thus, if the probe is detected on the support, coupledwith a lack of test compound binding, may indicate the test compound iscapable of binding to the CD36 binding domain.

Modulation is tested by screening for a test compound's ability tomodulate the activity of CD36 and includes combining a test compoundwith a CD36 binding domain, as described above, and determining analteration in the biological activity of the CD36. Therefore in thiscase, the test compound should both bind to the CD36 binding domain(although this may not be necessary), and alter its biological activityas defined herein.

Positive controls and negative controls may be used in the assays. Allcontrol and test samples are performed multiple times to obtainstatistically significant results. Following incubation, all samples arewashed free of non-specifically bound material and the amount of boundprobe determined. For example, where a radiolabel is employed, thesamples may be counted in a scintillation counter to determine theamount of bound compound.

Typically, the signals that are detected in the assay may includefluorescence, resonance energy transfer, time resolved fluorescence,radioactivity, fluorescence polarization, plasma resonance, orchemiluminescence and the like, depending on the nature of the label.Detectable labels useful in performing screening assays in thisinvention include a fluorescent label such as Fluorescein, Oregon green,dansyl, rhodamine, tetramethyl rhodamine, texas red, Eu³⁺; achemiluminescent label such as luciferase; colorimetric labels;enzymatic markers; or radioisotopes such as tritium, I¹²⁵ and the like.

Affinity tags, which may be useful in performing the screening assays ofthe present invention include biotin, polyhistidine and the like.

Additionally, the use of detectably labeled azapeptide compounds of thepresent invention is contemplated in a method for detecting the presenceor absence of CD36 in a biological sample, such as for example, a samplethat includes macrophages, endothelial cells or cardiomyocytes. Toachieve this, the sample would be contacted, either in vivo or in vitro,with the labeled compound. A signal from the labeled compound bound tothe CD36 would be measured, indicating the presence of CD36 in thesample.

Synthesis and Conformational Analysis of Azapeptide Analogs of GHRP-6

For the analysis of the conformational and structural requirements foraffinity and activity at the GHS-R1a and CD36 receptors, azapeptideanalogs were synthesized according to published methods (39, 40) usingFmoc-protected aza-amino acid chlorides to acylate the peptide chain.Removal of the Fmoc group and subsequent coupling of the next aminoacid, typically by way of the Fmoc-amino acid chloride, embeddedselectively the aza-amino acid residue within the peptide chain.

Analogues such as those presented in entries 12-70 of Table 1, haveprovided information concerning the active pharmacophore becauseaza-amino acid residues have been observed to stabilize turnconformations in model peptides (41). Moreover, these leads possessinteresting potential to exhibit enhanced durations of action, becauseazapeptides have previously exhibited resistance to enzymaticdegradation by proteases.

The replacement of Trp4 with azaPhe and with azaTyr improved selectivityby reducing affinity for the GHS-R1a and maintaining potency for theCD36 receptor. Peptide activity was similarly influenced on exchange ofTrp4 for other aza-aromatic residues (azaBip, azaNal and azahomoPhe),albeit with >10 fold reduction in potency at the CD36 receptor. Removalof the aromatic moiety by exchange of Trp4 for aliphatic aza-amino acidresidues (azaCha and azaLeu) diminished activity at both receptors. Theconformational bias of the aza-residue seems necessary for favoring sucharomatic interactions, because replacement of Trp4 by L- or D-Tyrdropped potency by ten-fold at the CD36 receptor. The loss in activityat GHS-R1a on replacing Trp4 with azaPhe is also consistent with earlierstudies that have suggested the indole NH of Trp4 may be implicated inreceptor binding of GHRP-6.

Replacement of D-Trp2 with aza-Tyr led to an analog with retainedpotency at the CD36 receptor. Similarly, swapping D-Trp2 for azaPhe,azaBip and for homo-azaPhe, all caused around a ten-fold drop at theCD36 receptor, and exchange of D-Trp2 for azaNal and for azaCha ledto >10 fold loss of potency at the CD36 receptor.

Replacement of Ala3 by azaGly and azaLeu has provided DBG188p, DBG201-A,CP-2B (i), CP-2B (ii), CP-2B (iii), and CP-2B (v), which retainedsignificant activity similar to the parent peptide. Moreover, inpreliminary data on ex vivo inhibition of neovascularization, theazaLeu3 analog DBG201-A exhibited the most potent inhibitory effect inaortic rings, better than GHRP-6.

Relatively, high activities for the azaTyr2, azaGly3, azaLeu3 andazaPhe4 analogs, all lead to the hypothesis that there are twosignificant pharmacophores at the ends of the peptide chain, which areoriented by the curvature of the chain. A turn centered at theD-Trp-Ala-Trp sequence is likely to be important for activity at CD36,in light of the facts that an aza-scan across this region has led to aseries of active candidates.

Peptides (compound numbers 32-33) and aza-peptides (compound numbers12-31 and 33-38) were prepared on solid support using an acid labileRink resin (42) and a Fmoc/tBu protocol (43). Incorporation of aza-aminoacid into peptide was performed using either 1,3,4-oxadiazol-2(3H)-onefor aza-Gly residue (44) or suitable N′-alkyl fluoren-9-ylmethylcarbazates to provide the corresponding Fmoc-aza amino acid chloride forcoupling onto the growing chain of the resin-bound peptide (39, 41).

Synthesis of Compound Numbers 12-38 and 66.68

Rink resin (0.65 mmol/g) was purchased from Advanced Chemtech Inc. andthe manufacturer's reported loading of the resin was used in thecalculation of the yields of the final products. 20% Phosgene in toluenewas purchased from Fluke. Melting points were uncorrected. ¹H and ¹³CNMR spectra were recorded respectively at 400 MHz and 100 MHz in CDCl₃or DMSO as the solvent and internal reference. Thin-layer chromatographywas performed on silica gel 60 F₂₅₄ plates from Merck. Flashchromatography was performed on silica gel 60 (230-400 Mesh ASTM) fromMerck. Analytical HPLC analyses were performed on a TARGA column fromHiggins Analytical, Inc. (4.6×250 mm, 5 μm, C₁₈) with a flow rate of 1.5mL/min using a 40 min linear gradient from water (0.1% TFA) to CH₃CN(0.1% TFA) (method 1) or MeOH (method 2). Retention times (Tr₁ and Tr₂)from analytical RP-HPLC are reported in minutes (Tr₁ for method 1, Tr₂for method 2). Peptides and aza-petides were purified usingsemi-preparative LC-MS (Previal 018 column, 22×250 mm², particle size 5μm) with solvent A, H₂O (0.1% TFA), and solvent B, acetonitrile (0.1%TFA) using a gradient of 20-40% of A over 20 min at a flow rate of 15mL/min.

9-H-Fluoren-9-ylmethyl carbazate (1)

To a well-stirred solution of hydrazine hydrate (19 g, 386 mmol) in 150mL of CH₃CN/H₂O (1/1, v/v), a solution of FmocCl (10 g, 38.65 mmol) in600 mL CH₃CN was added dropwise at 0° C. over 2 h. The reaction mixturewas then allowed to warm to room temperature and stirred for anadditional 12 hours, concentrated in vacuo to 150 mL and filtered toyield the title compound as a white solid, which was washed with waterand hexane and dried to a constant weight in vacuo (9.74 g, 99%): mp172-173° C.; ¹H NMR (DMSO) □4.08 (brs, 2H), 4.21 (t, J=7.2 Hz, 1H), 4.28(d, J=7.2 Hz, 2H), 7.32 (t, J=7.4 Hz, 2H), 7.42 (t, J=7.4 Hz, 2H), 7.69(d, J=7.4 Hz, 2H), 7.89 (d, J=7.4 Hz, 2H), 8.36 (brs, 1H); ¹³C NMR(DMSO) □□47.7, 66.7, 121.1 (2C), 126.3 (2C), 128.1 (2C), 128.7 (2C),141.7 (2C), 144.9 (2C), 159.2.

General procedure A for the synthesis of N′-alkyl fluorenylmethylcarbazates, 2-6

A suspension of 9-H-fluoren-9-ylmethyl carbazate 1 in EtOH (0.2 M) wastreated with 100 mol % of suitable aldehyde, heated at reflux for 2 hand concentrated in vacuo. The hydrazone was dissolved in THF (0.2 M)and treated successively with 110 mol % of AcOH and 110 mol % ofNaBH₃CN, stirred for 1 h and treated with additional NaBH₃CN ifnecessary until completion of the reaction was observed by TLC. Themixture was concentrated in vacuo. The residue was dissolved in EtOAc,washed with aqueous KHSO₄ (1M) and brine, dried over Na₂SO₄ andconcentrated under reduced pressure to yield a white solid, that wasdissolved in EtOH and heated at reflux for 1 h. The mixture wasconcentrated under reduced pressure to yield a residue that was isolatedby flash chromatography to yield the carbazate (2-6).

N′-Methyl-fluorenylmethyl carbazate (2)

Product from the reaction of carbazate 1 (7.1 mmol) and formaldehyde(7.1 mmol) was isolated in 77% yield by flash chromatography using a 30%EtOAc in hexane eluant as a white solid: Rf=0.54 (40% EtOAc in hexanes);mp 155-156° C.; ¹H NMR (DMSO) □2.43 (s, 3H), 4.22 (t, J=6.8 Hz, 1H),4.31 (d, J=6.8 Hz, 2H), 4.46 (brs, 1H), 7.32 (t, J=7.4 Hz, 2H), 7.41 (t,J=7.4 Hz, 2H), 7.69 (d, J=7.4 Hz, 2H), 7.88 (d, J=7.4 Hz, 2H), 8.67(brs, 1H); ¹³C NMR (DMSO) □□ 39.3, 47.7, 66.4, 121.1 (2C), 126.2 (2C),128.1 (2C), 128.7 (2C), 141.8 (2C), 144.9 (2C), 157.8, LRMS (EI) 179.2(M+H−100)⁺, 268.9 (M+H)⁺, 291.7 (M+Na)⁺; HRMS (EI) m/e for C₁₆H₁₇N₂O₂(M+H)⁺, calcd 269.1285. found 269.1291.

N′-Isopropyl-fluorenylmethyl carbazate (3)

Product from the reaction of carbazate 1 (3.9 mmol) and acetone (39mmol) was isolated in 68% yield by flash chromatography using a 30%EtOAc in hexane eluant as a white solid: Rf=0.20 (30% EtOAc in hexanes);mp 163-164° C.; ¹H NMR (DMSO) □0.91 (d, J=5.9 Hz, 6H), 2.51 (t, J=6.0Hz, 1H), 4.22 (t, J=6.6 Hz, 1H), 4.31 (d, J=6.6 Hz, 2H), 4.36 (brs, 1H),7.32 (t, J=7.4 Hz, 2H), 7.41 (t, J=7, 4 Hz, 2H), 7.71 (d, J=7.4 Hz, 2H),7.89 (d, J=7.4 Hz, 2H), 8.68 (brs, 1H); ¹³C NMR (DMSO) □□21.5 (2C),47.6, 50.3, 66.3, 121.0 (2C), 126.1 (2C), 127.9 (2C), 128.5 (2C), 141.6(2C), 144.7 (2C), 157.8. LRMS (EI) 296.9 (M+H)⁺. HRMS (EI) m/e forC₁₈H₂₁N₂O₂ (M+H)⁺, calcd 297.1598. found 297.1599.

N′-2-Isobutyl-fluorenylmethyl carbazate (4)

Product from the reaction of carbazate 1 (3.6 mmol) and 2-methylpropanal(3.6 mmol) was isolated in 72% yield by flash chromatography using a 20%EtOAc in hexane eluant as a white solid: Rf=0.30 (20% EtOAc in hexanes);mp 124-125° C.; ¹H NMR (DMSO) □0.87 (d, J=4.0 Hz, 6H), 1.63 (brs, 1H),2.50 (brs, 2H), 4.22 (t, J=6.3 Hz, 1H), 4.29 (d, J=6.3 Hz, 2H), 4.49(brs, 1H), 7.32 (t, J=7.4 Hz, 2H), 7.40 (t, J=7.4 Hz, 2H), 7.68 (d,J=7.4 Hz, 2H), 7.88 (d, J=7.4 Hz, 2H), 8.66 (brs, 1H); ¹³C NMR (DMSO)□□21.5 (2C), 27.1, 47.6, 59.6, 66.2, 121.0 (2C), 126.1 (2C), 127.9 (2C),128.5 (2C), 141.6 (2C), 144.7 (2C), 157.7. LRMS (EI) 310.9 (M+H)⁺. HRMS(EI) m/e for C₁₉H₂₃N₂O₂(M+H)⁺, calcd 311.1754. found 311.1761.

N′-cyclohexylmethyl-fluorenylmethyl carbazate (5)

Product from the reaction of carbazate 1 (2.5 mmol) and cyclohexanone(2.5 mmol) was isolated in 76% yield by flash chromatography using 50%EtOAc in hexane as eluant: white foam; Rf=0.26 (50% EtOAc in hexanes);¹H NMR (DMSO) □0.84 (q, J=11.2 Hz, 2H), 1.04-1.25 (m, 3H), 1.37 (m, 1H),1.54-1.70 (m, 3H), 1.75 (d, J=12.8 Hz, 2H), 2.53 (m, 2H), 4.22 (t, J=6.8Hz, 1H), 4.30 (d, J=6.8 Hz, 2H), 4.45 (m, 1H), 7.32 (t, J=8.4 Hz, 2H),7.42 (t, J=7.6 Hz, 2H), 7.70 (d, J=7.6 Hz, 2H), 7.89 (d, J=7.2 Hz, 2H),8.67 (m, 1H); ¹³C NMR (DMSO) □□25.7 (2C), 26.4 (2C), 31.0, 35.8, 46.8,57.5, 65.5, 120.2 (2C), 125.3 (2C), 127.1 (2C), 127.7 (2C), 140.8 (2C),143.9 (2C), 156.9. LRMS (EI) 351.1 (M+H)⁺, HRMS (EI) m/e for C₂₂H₂₇N₂O₂(M+H)⁺, calcd 396.1918. found 396.1919.

N′-2-phenylethyl-fluorenylmethyl carbazate (6)

Product from the reaction of carbazate 1 (2.5 mmol) andphenylacetaldehyde (2.5 mmol) was isolated in 76% yield by flashchromatography using 50% EtOAc in hexane as eluant: white foam; Rf=0.26(50% EtOAc in hexanes); ¹H NMR (DMSO) □2.67 (t, J=7.2 Hz, 2H), 2.94 (m,2H), 4.24 (t, J=6.8 Hz, 1H), 4.34 (d, J=6.8 Hz, 2H), 4.63 (m, 1H),7.14-7.36 (m, 7H), 7.42 (t, J=7.6 Hz, 2H), 7.71 (d, J=7.2 Hz, 2H), 7.90(d, J=7.6 Hz, 2H), 8.78 (brs, 1H); ¹³C NMR (DMSO) □□33.9, 46.8, 52.5,65.6, 120.2 (2C), 125.3 (2C), 125.9, 127.2 (2C), 127.8 (2C), 128.3 (2C),128.8 (2C), 140.2, 140.8 (2C), 143.9 (2C), 157.0. LRMS (EI) 359.1(M+H)⁺, 381.2 (M+Na)⁺. HRMS (EI) m/e for C₂₃H₂₃N₂O₂ (M+H)⁺, calcd396.1918. found 396.1919.

General procedure B for the synthesis of N′-alkyl fluorenylmethylcarbazates (7-11)

A suspension of 9-H-fluoren-9-ylmethyl carbazate 1 in EtOH (0.2 M) wastreated with 100 mol % of suitable aldehyde heated at reflux for 2 h,let cool and concentrated in vacuo. The hydrazone was dissolved in THF(0.2 M), treated with a suspension of 10 mol % of Pd(OH)₂ on carbon (20wt %) in THF, placed under H₂ gas at 100 psi and stirred at roomtemperature overnight. The reaction mixture was filtered over Celite.The filtrate was evaporated on a rotary evaporator. The N′-alkylfluorenylmethyl carbazate, 7-11 was isolated by flash chromatography.

N′-Benzyl-fluorenylmethyl carbazate (7)

Product from the reaction of carbazate 1 (7.8 mmol) and benzaldehyde(7.8 mmol) was isolated as a white solid in 94% yield by flashchromatography using a 30% EtOAc in hexane eluant: Rf=0.27 (30% EtOAc inhexanes); mp 143-145° C.; ¹H NMR (DMSO) □3.88 (brs, 2H), 4.21 (t, J=6.8Hz, 1H), 4.31 (d, J=6.8 Hz, 2H), 4.95 (brs, 1H), 7.15-7.35 (m, 7H), 7.41(t, J=7.4 Hz, 2H), 7.67 (d, J=7.6 Hz, 2H), 7.89 (d, J=7.6 Hz, 2H), 8.71(brs, 1H); ¹³C NMR (DMSO) □□47.7, 55.2, 66.4, 121.1 (2C), 126.3 (2C),127.9, 128.1 (2C), 128.7 (2C), 129.1 (2C), 129.5 (2C), 139.8, 141.8(2C), 144.8 (2C), 157.9. LRMS (EI) 344.9 (M+H)⁺, 688.9 (2M+H)⁺. HRMS(EI) m/e for C₂₂H₂₁N₂O₂ (M+H)⁺, calcd 345.1598. found 345.1603.

N′-(4-(tert-Butyldimethylsilyloxy))-benzyl)-fluorenylmethyl carbazate(8)

Product from the reaction of carbazate 1 (5.90 mmol) and4-(tert-Butyldimethylsilyloxy))-benzaldehyde (5.90 mmol) was isolated in70% yield as a colorless oil by flash chromatography using a 20% EtOAcin hexane eluant: Rf=0.30 (20% EtOAc in hexanes); ¹H NMR (DMSO) □0.18(s, 6H), 0.93 (s, 9H), 3.80 (brs, 2H), 4.20 (t, J=6.5 Hz, 1H), 4.29 (d,J=6.5 Hz, 2H), 4.83 (brs, 1H), 6.77 (d, J=8.3 Hz, 2H), 7.18 (d, J=8.0Hz, 2H), 7.31 (t, J=7.5 Hz, 2H), 7.41 (t, J=7.5 Hz, 2H), 7.67 (d, J=7.4Hz, 2H), 7.88 (d, J=7.5 Hz, 2H), 8.69 (brs, 1H); ¹³C NMR (DMSO) □□−3.5(2C), 19.0, 26.6 (3C), 47.7, 54.8, 66.5, 120.4 (2C), 121.1 (2C), 126.3(2C), 128.1 (2C), 128.7 (2C), 130.8 (2C), 132.4, 141.8 (2C), 144.8 (2C),155.0, 157.9. HRMS (EI) m/e for C₂₈H₃₅N₂O₃Si (M+H)⁺, calcd 475.2412.found 475.2414.

N′-(1-naphthylmethyl)-fluorenylmethyl carbazate (9)

Product from the reaction of carbazate 1 and 1-naphthylaldehyde wasisolated as a white solid in 25% yield by flash chromatography using 30%EtOAc in hexane as eluant: Rf=0.50 (40% EtOAc in hexanes); mp 136-137°C.; ¹H NMR (CDCl₃) □4.27 (t, J=6.7 Hz, 1H), 4.51 (m, 4H), 6.43 (bs, 1H),7.30-7.39 (m, 2H), 7.40-7.49 (m, 4H), 7.50-7.70 (m, 4H), 7.75-7.96 (m,4H), 8.31 (d, J=8.4 Hz, 1H); ¹³C NMR (CDCl₃) □□46.9, 53.1, 66.7, 119.7(2C), 123.6, 124.7 (2C), 125.0, 125.5, 126.1, 126.8 (2C), 127.5 (2C),127.6, 128.2, 128.3, 131.7, 132.4, 133.5, 141.0 (2C), 143.3 (2C), 156.9,LRMS (EI) 395.1 (M+H)⁺, 789.1 (2M+H)⁺. HRMS (EI) m/z for C₂₆H₂₃N₂O₂(M+H)⁺, calcd 395.1754. found 345.1755.

N′-(2-naphthylmethyl)-fluorenylmethyl carbazate (10)

Product from the reaction of carbazate 1 and 2-naphthylaldehyde wasisolated as a white solid in 50% yield by flash chromatography using 30%EtOAc in hexane as eluant: Rf=0.56 (40% EtOAc in hexanes); mp 128-129°C.; ¹H NMR (CDCl₃) □4.20 (m, 2H), 4.25 (t, J=6.8 Hz, 1H), 4.49 (m, 2H),6.45 (bs, 1H), 7.29-7.38 (m, 2H), 7.44 (t, J=7, 4 Hz, 2H), 7.48-7.55 (m,3H), 7.57-7.63 (m, 2H), 7.77-7.81 (m, 2H), 7.82-7.91 (m, 3H); ¹³C NMR(CDCl₃) □□46.8, 55.4, 66.7, 119.7 (2C), 124.7 (2C), 125.6, 125.8, 126.6,126.8 (2C), 127.4, 127.5 (2C), 127.5 (2C), 127.9, 132.6, 133.0, 134.5,141.0 (2C), 143.3 (2C), 156.9. LRMS (EI) 395.1 (M+H)⁺, 789.1 (2M+H)⁺,HRMS (EI) m/z for C₂₆H₂₃N₂O₂ (M+H)⁺, calcd 395.1754. found 345.1755.

N′-(biphenyl-4-ylmethyl)-fluorenylmethyl carbazate (11)

Product from the reaction of carbazate 1 and 4-biphenylaldehyde wasisolated as a white solid in 58% yield by flash chromatography using 30%EtOAc in hexane as eluant: Rf=0.35 (40% EtOAc in hexanes); mp 140-141°C.; ¹H NMR (CDCl₃) □4.09 (m, 2H), 4.27 (m, 1H), 4.51 (m, 2H), 6.44 (bs,1H), 7.30-7.52 (m, 9H), 7.55-7.68 (m, 6H), 7.80 (d, J=7.5 Hz, 2H)¹³C NMR(CDCl₃) □□46.5, 55.0, 66.6, 119.7 (2C), 124.7 (2C), 126.8 (4C), 126.9(2C), 127.0, 127.5 (2C), 128.5 (2C), 129.1 (2C), 136.0, 140.2, 140.4,141.0 (2C), 143.3 (2C), 156.8. LRMS (EI) 421.1 (M+H)⁺. HRMS (EI) m/z forC₂₈H₂₅N₂O₂ (M+H)⁺, calcd 421.1910. found 421.1910.

General Procedure for Fmoc Deprotection and HBTU Couplings

Peptide synthesis was performed under standard condition in an automatedshaker using Rink resin. Couplings of amino-acids (3 eq) were performedin DMF using HBTU (3 eq) as coupling reagent and DIEA (3 eq and 1 eq 20min after the start of the coupling reaction) as base. Fmocdeprotections were performed by treating resin with 20% piperidine inDMF for periods of 10 min and 20 min. Resin was washed after eachcoupling and deprotection step alternatively with DMF (2×), MeOH (2×)and DCM (2×).

General Procedure for Introduction of Aza-Amino Acid on Resin andCoupling of the Next Amino Acid

To a 0.1 M solution of a suitable N′-alkyl fluoren-9-ylmethyl carbazate(2-11) (300 mol % relative to resin loading) in dry DCM under argon at0° C., a solution of phosgene in toluene (20% by wt, 600 mol %) wasadded dropwise. After complete consumption of starting material (2-11)(usually after 15 min as indicated by TLC), the reaction mixture wasconcentrated in vacuo to yield the Fmoc-aza-amino acid chloride whichwas employed without further purification. The resulting Fmoc-aza-aminoacid chloride (300 mol %) was suspended in dry DCM (0.15 M), treatedwith DIEA (600 mol %) to obtain a clear solution, and transferred to avessel containing the resin-bound N-terminal amine swollen in dry DCM.The mixture was shaken overnight at room temperature under argon. Thesolution was filtered, the resin was washed twice with dry DCM and theaza-amino acid coupling procedure described above was repeated again.The resin was then treated under the conditions to remove the Fmoc groupthat were described above. The aza-amino acid resin was then swollen indry THF and treated with a solution of Fmoc-amino acid (300 mol %) inTHF (0.15 M), followed sequentially by BTC (100 mol %) and2,4,6-collidine (1400 mol %). The reaction mixture was shaken for 3 hunder argon and the resin was filtered. The resin was washedalternatively with DMF (2×), MeOH (2×) and DCM (2×) and peptidesynthesis was continued.

General Procedure for Side-Chain Deprotection and Aza-Peptide Cleavage

Aza-peptide resin was treated with a freshly made solution ofTFA/H₂O/TIS (95/2.5/2.5, v/v/v, 20 mL/g aza-peptide resin) for 2 h atroom temperature. The cleavage mixture was filtered and the resin waswashed with neat TFA. The filtrate was then concentrated to about 1 mLand treated with Et₂O. The resulting aza-peptide precipitate wasfiltered, washed with Et₂O and dissolved in an acetonitrile/H₂O (1/1,v/v) solution and lyophilized to yield a light foam or powder.

His-D-Trp-azaAla-Trp-D-Phe-Lys-NH₂ (12) Tr₁=14.85, Tr₂=22.03; LRMS (EI)calcd for C₄₅H₅₆N₁₃O₆ (M+H)⁺. 874.4 found m/e 874.3 (M+H)⁺,CH₃(CH₂)₄CO-D-Trp-azaAla-D-Phe-Lys-NH₂ (13) Tr₁=22.80, Tr₂=N.D.; LRMS(EI) calcd for C₄₅H₅₉N₁₀O₆ (M+H)⁺, 835.5. found m/e 835.4 (M+H)⁺.(CH₃)₂CHCH₂CO-D-Trp-azaAla-D-Phe-Lys-NH₂ (14) Tr₁=21.60, Tr₂=N.D.; LRMS(EI) calcd for C₄₃H₅₅N₁₀O₆ (M+H)⁺, 821.4. found m/e 821.4 (M+H)⁺.Ph-(CH₂)₂-CO-D-Trp-azaAla-Trp-D-Phe-Lys-NH₂ (15) Tr₁=22.77, Tr₂=N.D.;LRMS (EI) calcd for C₄₈H₅₇N₁₀O₆ (M+H)⁺, 869.4. found m/e 869.4 (M+H)⁺.CH₃CH(OH)—CO-D-Trp-azaAla-Trp-D-Phe-Lys-NH₂ (16) Tr₁=19.15, Tr₂=N.D.;LRMS (EI) calcd for C₄₂H₅₃N₁₀O₇ (M+H)⁺, 809.4. found m/e 809.4 (M+H)⁺.Ph-CO-D-Trp-azaAla-Trp-D-Phe-Lys-NH₂ (17) Tr₁=21.87, Tr₂=N.D.; LRMS (EI)calcd for C₄₆H₅₃N₁₀O₆(M+H)⁺, 841.4. found m/e 841.4 (M+H)⁺.Nal-1-CO-D-Trp-azaAla-Trp-D-Phe-Lys-NH₂ (18) Tr₁=23.71, Tr₂=N.D.; LRMS(EI) calcd for C₅₀H₅₅N₁₀O₆(M+H)⁺, 891.4. found m/e 891.4 (M+H)⁺.Cy-CO-D-Trp-azaAla-Trp-D-Phe-Lys-NH₂ (19) Tr₁=22.72, Tr₂=N.D.; LRMS (EI)calcd for C₄₆H₅₉N₁₀O₆(M+H)⁺. 847.5. found m/e 847.4 (M+H)⁺.In—CO-D-Trp-azaAla-Trp-D-Phe-Lys-NH₂ (2C) Tr₁=18.35/18.85, Tr₂=N.D.;LRMS (EI) calcd for C₄₉H₅₈N₁₀O₆ (M+H)⁺, 896.5. found m/e 896.4 (M+H)⁺.Ph-CH₂—CH(OH)—CO-D-Trp-azaAla-Trp-D-Phe-Lys-NH₂ (21) Tr₁=21.65,Tr₂=N.D.; LRMS (EI) calcd for C₄₈H₅₇N₁₀O₇ (M+H)⁺, 885.4. found m/e 885.4(M+H)⁺. His-azaTyr-Ala-Trp-D-Phe-Lys-NH₂ (22) Tr₁=13.41, Tr₂=20.21; LRMS(EI) calcd for C₄₃H₅₅N₁₂O₇ (M+H)⁺, 851.4. found m/e 851.3 (M+H)⁺.His-D-Trp-Ala-azaTyr-D-Phe-Lys-NH₂ (23) Tr₁=13.57, Tr₂=20.59; LRMS (EI)calcd for C₄₃H₅₅N₁₂O₇ (M+H)⁺, 851.4. found m/e 851.3 (M+H)⁺.His-D-Trp-azaGly-Trp-D-Phe-Lys-NH₂ (24) Tr₁=14.48, Tr₂=21.84; LRMS (EI)calcd for C₄₄H₅₄N₁₃O₆ (M+H)⁺. 860.4 found m/e 860.3 (M+H)⁺.His-D-Trp-azaLeu-Trp-D-Phe-Lys-NH₂ (25) Tr₁=16.24, Tr₂=25.33; LRMS (EI)calcd for C₄₈H₆₂N₁₃O₆ (M+H)⁺, 916.5. found m/e 916.5 (M+H)⁺.His-D-Trp-Ala-azaPhe-D-Phe-Lys-NH₂ (26) Tr₁=14.96, Tr₂=23.87; LRMS (EI)calcd for C₄₃H₅₅N₁₂O₆ (M+H)⁺, 835.4. found m/e 835.5 (M+H)⁺.His-D-Trp-Ala-azaLeu-D-Phe-Lys-NH₂ (27) Tr₁=14.27, Tr₂=22.83; LRMS (EI)calcd for C₄₀H₅₇N₁₂O₆ (M+H)⁺, 801.4. found m/e 801.5 (M+H)⁺.His-D-Trp-Ala-azaBip-D-Phe- Lys-NH₂ (28) Tr₁=17.81, Tr₂=27.68; LRMS (EI)calcd for C₄₉H₅₉N₁₂O₆ (M+H)⁺, 911.5. found m/e 911.5 (M+H)⁺.His-D-Trp-Ala-azaCha-D-Phe-Lys-NH₂ (29) Tr₁=16.08, Tr₂=25.73; LRMS (EI)calcd for C₄₃H₆₁N₁₂O₆(M+H)⁺, 841.5. found m/e 841.5 (M+H)⁺.His-D-Trp-Ala-azahomoPhe-D-Phe-Lys-NH₂ (30) Tr₁=15.44, Tr₂=24.69; LRMS(EI) calcd for C₄₄H₅₇N₁₂O₆ (M+H)⁺, 849.4. found m/e 849.5 (M+H)⁺.His-D-Trp-Ala-azaNal-1-D-Phe-Lys-NH₂ (31) Tr₁=16.53, Tr₂=26.08; LRMS(EI) calcd for C₄₇H₅₇N₁₂O₆ (M+H)⁺, 885.4. found m/e 885.5 (M+H)⁺.His-D-Trp-Ala-Tyr-D-Phe-Lys-NH₂ (32) Tr₁=12.59, Tr₂=20.64; LRMS (EI)calcd for C₄₄H₅₆N₁₁O₇ (M+H)⁺, 850.4. found m/e 850.5 (M+H)⁺.His-D-Trp-Ala-D-Tyr-D- Phe-Lys-NH₂ (33) Tr₁=13.17, Tr₂=20.99; LRMS (EI)calcd for C₄₄H₅₆N₁₁O₇ (M+H)⁺, 850.4. found m/e 850.5 (M+H)⁺.His-azaPhe-Ala-Trp-D-Phe-Lys-NH₂ (34) Tr₁=14.08, Tr₂=22.64; LRMS (EI)calcd for C₄₃H₅₅N₁₂O₆ (M+H)⁺, 835.4. found m/e 835.5 (M+H)⁺.His-azaNal-1-Ala-Trp-D-Phe-Lys-NH₂ (35) Tr₁=15.41, Tr₂=25.23; LRMS (EI)calcd for C₄₇H₅₇N₁₂O₆ (M+H)⁺, 885.4. found m/e 885.5 (M+H)⁺.His-azahomoPhe-Ala-Trp-D-Phe-Lys-NH₂ (36) Tr₁=14.80, Tr₂=23.37; LRMS(EI) calcd for C₄₄H₅₇N₁₂O₆ (M+H)⁺, 849.4. found m/e 849.5 (M+H)⁺.His-azaBip-Ala-Trp-D-Phe-Lys-NH₂ (37) Tr₁=16.88, Tr₂=26.80; LRMS (EI)calcd for C₄₉H₅₉N₁₂O₆ (M+H)⁺, 911.5. found m/e 911.5 (M+H)⁺.His-azaCha-Ala-Trp-D-Phe- Lys-NH₂ (38) Tr₁=14.93, Tr₂=24.75; LRMS (EI)calcd for C₄₃H₆₁N₁₂O₆ (M+H)⁺, 841.5. found m/e 841.5 (M+H)⁺.Phe-D-Trp-Ala-azaTyr-D-Phe-Lys-NH₂ (66) Tr₁=5.30^(d); LRMS (EI) calcdfor C₄₆H₅₆N₁₀O₇(M+H)⁺, 861.4. found m/e 861.3 (M+H)⁺, 883.3 (M+23)⁺,431.3 (M+2H)⁺. Ala-D-Trp-Ala-azaTyr-D-Phe-Lys-NH₂ (67) Tr₁4.82; LRMS(EI) calcd for C₄₀H₅₂N₁₀O₇(M+H)⁺, 785.4. found m/e 785.3 (M+H)⁺, 807.2(M+23)⁺, 393.4 (M+2H)⁺. Hydrocinnamyl-D-Trp-Ala-azaTyr-D- Phe-Lys-NH₂(68) Tr₁=5.04^(e); LRMS (EI) calcd for C₄₆H₅₅N₉O₇ (M+H)⁺, 846.4. foundm/e 846.5 (M+H)⁺, 868.4 (M+23).

Peptide Synthesis

Peptides were synthesized on Rink resin (0.65 mmol/g) under standardconditions (43) in an automated shaker. Couplings of Fmoc-amino acids(300 mol %) were performed in DMF using HBTU (300 mol %) as couplingreagent and DIEA (300 mol %) as base. Side-chains for Lys and D-Trp wereprotected by Boc group and His with a trityl group. Fmoc deprotectionswere performed by treating resin with 20% piperidine in DMF for periodsof 10 min and 20 min. Resin was washed after each coupling anddeprotection step alternatively with DMF (2×), MeOH (2×) and DCM (2×).Side-chain deprotection and peptide cleavage were performed with afreshly made solution of TFA/H₂O/TIS (95/2.5/2.5, v/v/v, 20 mL/g peptideresin) for 2 h at room temperature. The cleavage mixture was filteredand the resin was washed with neat TFA. The filtrate was thenconcentrated and treated with chilled Et₂O to precipitate the peptides.The peptides were removed by centrifugation, dissolved in CH₃CN/H₂O(1/1, v/v) and lyophilized. The crude material was purified bysemi-preparative RP-HPLC (Higgins C18 column, 20×250 mm, particle size 5μm) with solvent A, H₂O (0.1% TFA) and solvent B, CH₃CN (0.1% TFA).Analytical HPLC condition I was performed on a Xterra MS C18 column(4.6×150 mm, particle size 5 μm) using a gradient of 0-65% eluant B in Aover 20 min with a flow rate of 0.5 mL/min and the detector centered at210 nm: B (CH₃CN) in A (H₂O containing 0.1% formic acid) and retentiontimes (t_(R)) are reported in minutes. Analytical HPLC condition II wasperformed on a Xterra MS C18 column (4.6×150 mm, particle size 5 μm)using a gradient of 0-65% eluant B (MeOH) in A (H₂O containing 0.1%formic acid) over 20 min with a flow rate of 0.5 mL/min.

Synthesis of Compound Numbers 39-65 General procedure for the synthesisof N′-Alkyl-fluorenylmethyl carbazates

N′-2-Isobutyl-fluorenyl methyl carbazate, N′-benzyl-fluorenylmethylcarbazate, and N′-(4-(t-butyldimethylsilyloxy)-benzyl)-fluorenyl-methylcarbazate as azapeptide precursors were synthesized according topublished procedure (39).

Fluoren-9-yl-methyl pyrazolidine-1-carboxylate hydrochloride

Tert-butyl pyrazolidine-1-carboxylate (39) (1.39 g, 8.08 mmoles) wasacylated with Fmoc succinimide (3.27 g, 9.69 mmoles, 1.2 eq.) in drydichloromethane (20 mL). The reactive mixture was left stirringovernight. The volatiles were removed using a rotary evaporator and theresidue was dissolved in EtOAc. The organic layer was extracted threetimes, respectively, with 5% citric acid, 5% NaHCO₃ and brine, driedover Na₂SO₄, filtered and concentrated to a white foam which waspurified by column chromatography using 7:3 EtOAc:Hexane as eluant.Evaporation of the collected fractions afforded1-(9H-fluoren-9-yl)methyl 2-tert-butyl pyrazolidine-1,2-dicarboxylate asa white solid in a 94% yield. Pyrazolidine-1,2-dicarboxylate (1.34 g,3.4 mmoles) was treated with 25 mL of a 1:1 TFA:DCM solution and stirredfor one hour. Removal of the volatiles by rotary evaporation gave aresidue that was dissolved in 1N HCl, stirred for 1 hour andfreeze-dryed to yield hydrochloride(9H-fluoren-9-yl)methyl-1-carboxylate: mp (143.2-148.1° C.), ¹H NMR(DMSO) δ 7.89 (d, J=7.32 Hz, 2H), 7.72 (d, J=7.33 Hz, 2H), 7.43 (ddd,J=7.36 Hz, 0.57 Hz, 2H), 7.33 (ddd, J=7.42 Hz, 1.14 Hz, 2H), 4.44 (d,J=6.90 Hz, 2H), 4.33 (t, J=6.56 Hz, 1H), 3.58 (t, J=6.96 Hz, 2H), 3.43(t, J=6.96 Hz, 2H), 2.20 (m, 2H). ¹³C (DMSO) δ 154.57, 144.64, 142.11,129.26, 126.54, 121.67, 69.89, 47.50, 25.50.

Solid-Phase Azapeptide Synthesis

Incorporation of aza-amino acids on resin was performed according toliterature procedure (39). To generate a library of 29 azapeptides inparallel, IRORI kan technology was employed. Macrokans were respectivelyfilled with 130 mg (0.0845 mmole) of 75-100 mesh Rink Resin SS and aradiofrequency (Rf) tag associated to a unique ID number. In asplit-and-mix approach, kans undergoing identical reactions were pooledtogether in a normal glass vessel, that was filled with solvent andreagents which difused through the outer mesh walls of themicroreactors. Upon completion of the reaction, Macrokans wereseparated, sorted, and pooled accordingly for next reactions (39).

His-azaPhe-Ala-Trp-D-Phe-Ala-NH₂ (39) Tr₁=15.54, Tr₂=11.05; LRMS (EI)calcd for C₄₀H₄₇N₁₁O₆(M+H)⁺, 778.4 found m/e 778.4 (M+H)⁺, 800.4(M+23)⁺. His-azaPhe-Ala-Trp-D-Ala-Lys-NH₂ (40) Tr₁=9.08, Tr₂=9.11; LRMS(EI) calcd for C₃₇H₅₀N₁₂O₆ (M+H)⁺, 759.4. found m/e 759.4 (M+H)⁺, 781.4(M+23)⁺, (D/L)His-azaPhe-Ala-Ala-D-Phe-Lys-NH₂ (41) Tr₁=9.99, Tr₂=11.93;LRMS (EI) calcd for C₃₅H₄₉N₁₁O₆(M+H)⁺, 720.4. found m/e 720.4 (M+H)⁺.Ala-azaPhe-Ala-Trp-D-Phe-Lys-NH₂ (42) Tr₁=15.67, Tr₂=7.80^(c); LRMS (EI)calcd for C₄₀H₅₂N₁₀O₆ (M+H)⁺, 769.4. found m/e 769.4 (M+H)⁺.His-azaTyr-Ala-Trp- D-Phe-Ala-NH₂ (43) Tr₁=14.89, Tr₂=21.34; LRMS (EI)calcd for C₄₀H₄₇N₁₁O₇(M+H)⁺, 794.4. found m/e 794.4 (M+H)⁺, 816.4(M+23)⁺. His-azaTyr-Ala-Trp-D-Ala-Lys-NH₂ (44) Tr₁=8.79, Tr₂=10.79; LRMS(EI) calcd for C₃₇H₅₀N₁₂O₇ (M+H)⁺, 775.4. found m/e 775.4 (M+H)⁺, 797.4(M+23)⁺. (D/L)His-azaTyr-Ala-Ala-D-Phe-Lys-NH₂ (45) Tr₁=8.13, Tr₂=8.18;LRMS (EI) calcd for C₃₅H₄₉N₁₁O₇(M+H)⁺, 736.4. found m/e 736.4 (M+H)⁺,758.4 (M+23)⁺. Ala-azaTyr-Ala-Trp- D-Phe-Lys-NH₂ (46) Tr₁=13.19,Tr₂=19.02; LRMS (EI) calcd for C₄₀H₅₂N₁₁O₇(M+H)⁺, 785.4. found m/e 785.4(M+H)⁺, 807.4 (M+23)⁺. His-D-Trp-azaGly-Trp-D-Phe-Ala-NH₂ (47)Tr₁=11.01^(b), Tr₂=18.56; LRMS (EI) calcd for C₄₁H₄₆N₁₂O₆ (M+H)⁺, 803.4.found m/e 803.4 (M+H)⁺, 825.4 (M+23)⁺,His-D-Trp-azaGly-Trp-D-Ala-Lys-NH₂ (48) Tr₁=9.95, Tr₂=12.54; LRMS (EI)calcd for C₃₈H₄₉N₁₃O₆(M+H)⁺, 784.4. found m/e 784.4 (M+H)⁺.His-D-Trp-azaGly-Ala-D-Phe-Lys-NH₂ (49) Tr₁=8.88, Tr₂=8.95; LRMS (EI)calcd for C₃₆H₄₈N₁₂O₆ (M+H)⁺, 745.4. found m/e 745.4 (M+H)⁺, 767.4(M+23)⁺. His-D-Ala-azaGly-Trp-D-Phe-Lys-NH₂ (50) Tr₁=9.64, Tr₂=9.80;LRMS (EI) calcd for C₃₆H₄₈N₂O₆ (M+H)⁺, 745.4. found m/e 745.4 (M+H)⁺.Ala-D-Trp-azaGly-Trp-D-Phe-Lys-NH₂ (51) Tr₁=15.61, Tr₂=17.95^(b); LRMS(EI) calcd for C₄₁H₅₁N₁₁O₆(M+H)⁺, 794.4. found m/e 794.4 (M+H)⁺.His-D-Trp-azaLeu-Trp-D-Phe-Ala-NH₂ (52) Tr₁=17.54, Tr₂=13.64; LRMS (EI)calcd for C₄₅H₅₄N₁₂O₆(M+H)⁺, 859.4. found m/e 859.4 (M+H)⁺, 881.4(M+23)⁺. His-D-Trp-azaLeu-Trp-D-Ata-Lys-NH₂ (53) Tr₁=11.65, Tr₂=11.73;LRMS (EI) calcd for C₄₂H₅₇N₁₃O₆(M+H)⁺, 840.5. found m/e 840.5 (M+H)⁺,862.4 (M+23)⁺. His-D-Trp-azaLeu-Ala-D-Phe-Lys-NH₂ (54) Tr₁=14.55,Tr₂=12.10; LRMS (EI) calcd for C₄₀N₅₆N₁₂O₆(M+H)⁺, 801.5. found m/e 801.5(M+H)⁺. His-D-Ala-azaLeu-Trp-D-Phe-Lys-NH₂ (55) Tr₁=11.16, Tr₂=10.60b;LRMS (EI) calcd for C₄₀H₅₆N₁₂O₆(M+H)⁺, 801.4. found m/e 801.4 (M+H)⁺,823.4 (M+23)⁺. Ala-D-Trp-azaLeu-Trp-D-Phe-Lys-NH₂ (56) Tr₁=17.08,Tr₂=12.08^(c); LRMS (EI) calcd for C₄₅H₅₉N₁₁O₆ (M+H)⁺, 850.5. found m/e850.5 (M+H)⁺, 872.5 (M+23)⁺. His-D-Trp- Ala-azaPhe-D-Phe-Ala-NH₂ (57)Tr₁=16.55, Tr₂=11.96; LRMS (EI) calcd for C₄₀H₄₇N₁₁O₆ (M+H)⁺, 778.4.found m/e 778.4 (M+H)⁺, 800.4 (M+23)⁺.His-D-Trp-Ala-azaPhe-D-Ala-Lys-NH₂ (58) Tr₁=10.00, Tr₂=10.10; LRMS (EI)calcd for C₃₇H₅₀N₁₂O₆ (M+H)⁺, 759.4. found m/e 759.4 (M+H)⁺. 781.4(M+23)⁺. His-D-Ala-Ala-azaPhe-D-Phe-Lys-NH₂ (59) Tr₁=8.43, Tr₂=9.97;LRMS (EI) calcd for C₃₇H₄₉N₁₁O₆(M+H)⁺, 720.4. found m/e 720.4 (M+H)⁺,742.4 (M+23)⁺. Ala-D-Trp-Ala- azaPhe-D-Phe-Lys-NH₂ (60) Tr₁=15.86,Tr₂=10.62; LRMS (EI) calcd for C₄₀H₅₂N₁₀O₆(M+H)⁺, 769.4. found m/e 791.4(M+23)⁺. His-D-Trp-azaGly-Pro-D-Phe-Lys-NH₂ (61) Tr₁=8.90, Tr₂=11.04;LRMS (EI) calcd for C₃₈H₅₀N₁₂O₆(M+H)⁺, 771.4. found m/e 771.4 (M+H)⁺,793.4 (M+23)⁺. His-D-Trp-azaPro-Trp-D-Phe-Lys-NH₂ (62) Tr₁=12.85,Tr₂=17.73; LRMS (EI) calcd for C₄₆H₅₅N₁₃O₆(M+H)⁺, 900.5. found m/e 900.5(M+H)⁺. His-D-Trp-Ala-azaPro-D-Phe-Lys-NH₂ (63) Tr₁=10.94, Tr₂=10.95;LRMS (EI) calcd for C₃₉H₅₂N₁₂O₆(M+H)⁺, 785.4. found m/e 785.4 (M+H)⁺.807.4 (M+23)⁺. His-D-Trp- Ala-Trp-azaPro-Lys-NH₂ (64) Tr₁=9.96,Tr₂=12.61; LRMS (EI) calcd for C₄₀H₅₁N₁₃O₆(M+H)⁺, 824.4. found m/e 824.4(M+H)⁺, 846.4 (M+23)⁺. (D/L)His-azaPro-Ala-Trp-D-Phe-Lys-NH₂ (65)Tr₁=9.79, Tr₂=9.82; LRMS (EI) calcd for C₃₈H₅₀N₁₂O₆(M+H)⁺, 785.4. foundm/e 785.4 (M+H)⁺, 807.4 (M+23)⁺.

^(a) Unless otherwise noted, analytical HPLC analyses were performed ona 5 μM 150 mm×4.6 mm C18 Gemini column with a flow rate of 0.5 ml/minusing a 2-40 gradient from water (0.1% FA) to CH₃CN (0.1% FA) or MeOH(0.1% FA). ^(b) Analytical HPLC analyses were performed using the samecolumn as in a, with a 10-50 gradient from water (0.1% FA) to CH₃CN(0.1% FA) or MeOH (0.1% FA). ^(c) Analytical HPLC analyses wereperformed using the same column as in a, with a 20-80 gradient fromwater (0.1% FA) to CH₃CN (0.1% FA) or MeOH (0.1% FA). ^(d) AnalyticalHPLC analyses were performed using the same column as in a, with a 0-60gradient from water (0.1% FA) to CH₃CN (0.1% FA). ^(e) Analytical HPLCanalyses were performed using the same column as in a, with a 10-80gradient from water (0.1% FA) to CH₃CN (0.1% FA).

Compound MS Entry# # Structure (M + 1) 12 DBG-145p

874.3 13 DBG-168-1

835.4 14 DBG-168-2

821.4 15 DBG-168-3

869.4 16 DBG-168-4

809.4 17 DBG-168-5

841.4 18 DBG-168-6

891.4 19 DBG-168-7

847.4 20 DBG-168-8p

896.4 21 DBG-168- 10p

885.4 22 DBG-175p

851.3 23 DBG-178p

851.3 24 DBG-188p

860.3 25 DBG-201-A

26 DBG-253-1

835.5 27 DBG-253-2

801.5 28 DBG-253-3

911.5 29 DBG-253-4

841.5 30 DBG-253-5

849.5 31 DBG-253-6

885.5 32 DBG-253-7

850.5 33 DBG-253-8

850.5 34 DBG-253-9

835.5 35 DBG-253-10

885.5 36 DBG-253-11

849.5 37 DBG-253-12

911.5 38 DBG-253-13

841.5 39 CP-1A(i)

778.4 40 CP-1A(ii)

759.4 41 CP-1A(iii)

720.4 42 CP-1A(iv)

769.4 43 CP-1B(i)

794.4 44 CP-1B(ii)

775.4 45 CP-1B(iii)

736.4 46 CP-1B(iv)

785.4 47 CP-2A(i)

803.4 48 CP-2A(ii)

784.4 49 CP-2A(iii)

745.4 50 CP-2A(iv)

745.4 51 CP-2A(v)

794.4 52 CP-2B(i)

859.4 53 CP-2B(ii)

840.5 54 CP-2B(iii)

801.5 55 CP-2B(iv)

801.5 56 CP-2B(v)

850.5 57 CP-3(i)

778.4 58 CP-3(ii)

759.4 59 CP-3(iii)

720.4 60 CP-3(iv)

769.4 61 CP- Azagly_Pro

771.4 62 CP-AP-4

900.5 63 CP-AP-3

785.4 64 CP-AP-2

824.4 65 CP-AP-5

785.4 66 ZS554-F29

861.3 67 ZS555-F40

785.3 68 ZS556

846.5 69 DS85C

913.5 and 916.2 70 DS86D

900.4 ^(a)Analytical HPLC analyses were performed on a TARGA column fromHiggins Analytical, Inc. (4.6 × 250 mm, 5 μm, C₁₈) with a flow rate of1.5 mL/min. using a 40 min linear gradient from water (0.1% TFA) toCH₃CN (0.1% TFA). ^(b)HPLC purity at 214 nm of the crude peptide^(c)HPLC purity at 214 nm of the purified peptide ^(d)Crude yieldaccording to manufacturer's reported loading of the rink resin^(e)Yields after purification by RP-HPLC are based on manufacturer'sreported loading for Rink resin.

The yields and purities of compounds 39-65 are illustrated in Table 2.

TABLE 2 Yields and Purities of compounds 39-65 Item/ T_(R) T_(R) HPLCEntry (min) in (min) in purity at Yield Expected No. ACN^(a) MEOH 214nm^(d) (%)^(f) Mass Mass^(g) 39 15.54 11.05 96.8% 4.38 778.37108778.37835, 800.36030 40 9.08 9.11  >99% 1.15 759.39763 759.40490,781.38685 41 9.99 11.93 1:1 3.95 720.39400 720.39371 mixture, >99%^(e)42 15.67 7.80^(c)   93% 6.39 769.41440 769.41370 43 14.89 21.34   98%3.72 794.36599 794.37327, 816.35521 44 8.79 10.79   97% 4.90 775.39254775.39982, 797.38176 45 8.13 8.18 1:3 12.5 736.38164 736.38892, mixture,758.37086  >99%^(e) 46 13.19 19.02  >99% 3.65 785.40932 785.40858,807.38983 47 11.01^(b) 18.56^(b)   99% 4.98 803.36633 803.37360,825.35555 48 9.95 12.54  >99% 1.60 784.39288 784.40015 49 8.88 8.95 >99% 6.16 745.38198 745.38925, 767.37120 50 9.64 9.80  >99% 4.59745.38198 745.38823 51 15.61 17.95^(b)   99% 6.79 794.40238 794.40965 5217.54 13.64^(c)  >99% 3.83 859.42893 859.43620, 881.41815 53 11.65 11.73 >99% 7.22 840.45548 840.46275, 862.44470 54 14.55 12.10  >99% 3.57801.45185 801.45061 55 11.16 10.60^(b)  >99% 3.28 801.44458 801.45185,823.43380 56 17.08 12.08^(c)  >99% 4.26 849.46498 850.47226, 872.4542057 16.55 11.96  >99% 9.19 778.37108 778.37835, 800.36030 58 10.00 10.10 >99% 1.72 759.39763 759.40406, 781.38696 59 8.43 9.97  >99% 8.50720.39400 720.39526, 742.37676 60 15.86 10.62^(c)  >99% 6.86 769.40713791.39635 61 8.90 11.04  >99% 2.64 771.39763 771.40490, 793.38685 6212.85 17.73  >99% 2.26 900.45548 900.46164 63 10.94 10.95  >99% 2.02785.42055 785.42010, 807.39584 64 9.96 12.61  >99% 1.00 824.43145824.43075, 846.41257 65 9.79 9.82  >99%, 1.00 785.42055 785.42159,mixture^(e) 807.39807 ^(a)Unless otherwise noted, analytical HPLCanalyses were performed on a 5 μM 150 mm × 4.6 mm C18 Gemini column witha flow rate of 0.5 ml/min using a 2-40 gradient from water (0.1% FA) toCH₃CN (0.1% FA) or MeOH (0.1% FA). ^(b)Analytical HPLC analyses wereperformed using the same column as in a, with a 10-50 gradient fromwater (0.1% FA) to CH₃CN (0.1% FA) or MeOH (0.1% FA). ^(c)AnalyticalHPLC analyses were performed using the same column as in a, with a 20-80gradient from water (0.1% FA) to CH₃CN (0.1% FA) or MeOH (0.1% FA),^(d)HPLC purity at 214 nm of the purified peptide. ^(e)Mixture ofisomers shows up as two distinct peaks with identical masses. ^(f)Yieldsafter purification by HPLC are based on manufacturer's reported loadingfor Rink resin. ^(g)Observed masses corresponding to the H⁺ and Na⁺adducts.CD36 Receptor Covalent Binding Assay

For the determination of binding affinity of azapeptide derivatives ofGHRP-6 towards the scavenger receptor CD36, competition curves were setusing rat cardiac membrane preparation as source of CD36 andphotoactivatable derivative [¹²⁵I]Tyr-Bpa-Ala-hexarelin as covalentphotolabelling tracer as reported previously (45). Azapeptidederivatives (DBG series) were used as competition ligands atconcentrations ranging from 10⁻⁷ to 5.10⁻⁵ M. The IC₅₀s determined fromthe competition curves for the DBG compounds tested and that ofhexarelin, the GHRP prototype reported as ligand of CD36 (38) arecompiled in Table 3.

According to the structure-binding activity relationship of thecompounds tested, favoring the interaction of two aromatic constituentsat the ends of the peptide chain oriented by the curvature of the chainis essential for the selective binding affinity towards CD36. Forexample, the replacement of the Trp4 residue of GHRP-6 by azaPhe orazaTyr improved the selectivity by reducing the binding affinity forGHS-R1a receptor and maintaining the binding activity to CD36 (DBG253-1and DBG 178).

Methodology for the CD36 Covalent Photolabelled Binding Assay

Cardiac Membrane Preparation.

The hearts from Sprague Dawley rats (300 g-325 g) from Charles River,used as a source of cardiac membranes, were prepared as follows: theheart was washed with ice-cold saline, and fatty and connective tissuewas removed. The tissue was cut into small pieces with scissors andplaced in a Sorvall centrifuge tube containing 5 ml/g of fresh tissue ofBuffer A (10 mM NaHCO₃, 5 mM NaN₃, 10 μM Pefabloc, 0.1 μM Aprotinin, 1μM Pepstatin A, 1 μM Leupeptin, pH 7.0). The suspension was homogenizedwith a Polytron at low speed, 3×15 sec. The homogenate was centrifugedat 8 700×g for 20 min and the supernatant was put on ice. The pellet wasresuspended and homogenized with a Glass-teflon Potter by 5 strokes. Thehomogenate was centrifuged at 8 700×g for 10 min and this supernatantwas combined with the first supernatant. The supernatant fluid wascentrifuged at 35 000×g for 20 min, yielding a second pellet fractionthat was suspended thoroughly in a glass homogenizer with a Teflonpestle in 20 ml/g of fresh tissue of Buffer B (20 mM Tris-maleate, 0.6MKCl, pH 6.8). The resulting suspension was centrifuged again at 35 000×gfor 60 min. The third pellet fraction was resuspended thoroughly in aglass homogenizer with a Teflon pestle in 20 ml/g of fresh tissue ofBuffer C (10 mM Tris/HCl buffer pH 7.4) followed with anothercentrifugation at 35 000×g for 60 min. The harvested precipitate wasthen suspended in a small volume (1-2 ml/g of fresh tissue) of 50 mMTris/HCl buffer pH 7.4) containing 2 mM EGTA. The resulting membranepreparation was frozen at −80° C. Protein concentration was determinedwith bicinchoninic acid (BCA) method, using BSA as standard.

Radiolabelling of the Tracer

The iodination procedure was performed in the darkness.Tyr-Bpa-Ala-Hexarelin (10 nmol) was mixed with 100 ng of lactoperoxidaseand 1 mCi of Na¹²⁵I in a volume of 30 ul of 0.1N sodium acetate bufferpH 5.6. The reaction was started by adding 5 ul (3 nmol) of H₂O₂. Theincubation was carried out at 22° C. for 5 min. The addition of H₂O₂ wasrepeated twice with 5 min incubation each time. The reaction was stoppedwith the addition of 1.4 ml of 0.1% TFA. The iodinated peptide waspurified on a reverse-phase Vydac C₁₈ column with a 60 min lineargradient (1 ml/min) from 20% to 50% acetonitrile (90%) in 0.1% TFA(10%).

Competition Binding Curves

The receptor binding assays of the photoactivatable ligand wereperformed as follows: Membranes (200 ug/75 ul) were incubated in thedarkness, in 525 ul of 50 mM Tris-HCl pH 7.4 containing 2 mM EGTA(Buffer A) in the presence of a fixed concentration of[¹²⁵I]-Tyr-Bpa-Ala-Hexarelin (750 000 cpm/75 ul) in Buffer B (50 mMTris-HCl pH 7.4 containing 2 mM EGTA and 0.05% Bacitracin) and ofincreasing concentrations of DBG derivatives ranging from 0.1 to 50 μMas competition ligands. Nonspecific binding was defined as binding notdisplaced by 50 μM corresponding peptide. All peptide containingsolutions were diluted in Buffer B. Buffer A and B were degassed undervacuum, and used in capped tubes in order to minimize lipidperoxydation. Before the incubation period, all tubes were put under alow flow of nitrogen. All material was kept on ice and the binding assaywas performed in darkness. After an incubation period of 60 min at 22°C. (vortexing every 15 min), membranes were submitted to irradiationwith UV lamps (365 nm) for 15 min at 4° C. After centrifugation at 12000×g for 15 minutes, the pellets were resuspended in 150 μl of samplebuffer (62 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 15%2-mercapto-ethanol, and 0.05% bromophenol blue), and boiled for 5 minprior to being subjected to electrophoresis. Proteins (200 μg/100 μl)were separated on 7.5% SDS-PAGE (45 V overnight). The gels resultingfrom SDS/PAGE were fixed, colored in Coomassie Brilliant Blue R-250,dried, exposed to a storage phosphor intensifying screen (AmershamBiosciences), and analysed by using a Typhoon PhosphorImager (AmershamBiosciences) and ImageQuant 5.0 software to establish competitioncurves. Protein bands were quantified by densitometry. The covalentbinding signal of 87 kDa was analyzed by densitometry using TyphoonPhospholmager™ (Typhoon, Amersham Biosciences) and ImageQuant 5.0®software to set competition curves.

Specific binding activities were defined as the ratio of the density ofthe protein band to that of a non-specific band. Results are expressedas the percentage of the density of the pro to that of the total bindingband.

GHS-R1a Binding Assay

The binding affinity of the azapeptide derivatives of GHRP-6 towards theghrelin receptor GHS-R1a was documented by competition binding studieson the membranes of LLCPK-1 overexpressing GHS-R1a source of receptorand [¹²⁵I]-ghrelin (1-28) as radioligand (46). The azapeptidederivatives were used as competition ligands ranging from 10⁻¹² to 10⁻⁶M. The IC₅₀s of the competition binding curves for these derivatives andthat of GHRP-6 and (1-28) ghrelin as natural ligand for GHS-R1a arecompiled in Table 3 below. The replacement by aza amino acid residueswithin the structure of GHRP-6 reduced significantly the bindingaffinity towards GHS-R1a receptor at least of two decades as compared tothat of GHRP-6 and ghrelin which were in the subnanomolar to nanomolarranges.

Methodology for GHS-R1a Binding Assay

Radioiodination of Ghrelin

Rat ghrelin was radio iodinated with Na¹²⁵I using the lodo-Beadsiodination reagent (Pierce #28665). Briefly, an lodo-bead was incubatedwith 2 mCi of Na¹²⁵I (Amersham) in 100 μL of 50 mM sodium phosphatebuffer, pH 7.0 for 5 min at 22° C. The reaction was started by adding 20nmol of the peptide (1 mM in 0.1 M acetic acid). The tube was incubatedfor 10 min at 22° C. The iodination was terminated by adding 1 mL of0.1% TFA. The mono-iodinated peptide was purified by reverse-phase HPLCusing a Vydac C₁₈ (5 μM) column (30 cm×0.39 cm) in combination with abinary elution system of (A) 0.1% TFA in H₂O and (B) 0.1% TFA, 10% H₂O,90% acetonitrile. Fractions of 0.5 mL were collected and analyzed by itsradioactive content.

Expression of Human GHS-R1a in LLC-PK1 Cells

LLC-PK1 cells (ATCC #CL-101) were grown in Dulbecco's modified Eagle'smedium (Invitrogen) supplemented with 10% fetal bovine serum(Invitrogen) and 100 units of penicillin/streptomycin (Invitrogen) in a5% CO₂ incubator at 37° C. Cells (1.4×10⁶) were plated in 100 mm platesand transfected 24 h later with pCDNA3.1-hGHS-R1a (UMR cDNA ResourceCenter #GHSR0A0000) following the CaHPO₄ precipitation method. Briefly,40 μg of plasmid DNA was diluted in 500 μL of sterile H₂O and added toan equal volume of a 4× CaCl₂, pH 7.9 solution (2 mM Tris-HCl, 0.2 mMEDTA, 500 mM CaCl₂). The mixture was then added slowly with air bubblesto 1 mL of 2× Hepes-buffered saline, pH 7.1 (50 mM Hepes, 280 mM NaCl,1.5 mM Na₂HPO₄). After 30 min at room temperature, 1 mL was distributedevenly over the cells and let overnight. The medium was changed the nextmorning and the membrane preparation was made two days later.

Membrane Preparation of Transfected LLC-PK1 Cells

Cells were washed twice with PBS and once with ice-cold homogenizationbuffer (50 mM Tris-HCl, pH 7.3, 5 mM MgCl₂, 2.5 mM EDTA, 30 μg/mlbacitracin). Cells were scraped and harvested in 1.5 mL tubes. Cellswere then lysed by temperature shock with two freeze/thaw cycles inliquid nitrogen. The tubes were centrifuged at 14000 g for 20 min.Pellets were resuspended in homogenization buffer and stored at −80° C.until used. Total protein concentrations were determined with the BCAprotein assay kit (Pierce).

Receptor Binding Assay with ¹²⁵I-Ghrelin

Competition experiments were performed by incubating 50 μg of LLC-PK1membranes expressing human GHS-R1a with 15 fmol of ¹²⁵I-Ghrelin andincreasing concentrations of unlabeled ghrelin or with increasingconcentrations of azapeptides derivatives in 0.5 mL of binding buffer.Bound radiolabelled ligand was separated from free by filtration onWhatman GF/C filters pretreated with a 1% polyethylenimine solution.Filters were washed with 2×3 mL of washing buffer (50 mM Tris-HCl, pH7.3, 10 mM MgCl₂, 2.5 mM EDTA, 0.015% Triton X-100). Filters werecounted in a LKB gamma counter. Curves were analyzed using Prismsoftware (GraphPad Software Inc).

Inhibition of Microvascular Sprouting from Aortic Explants

To evaluate the antiangiogenic property of azapeptide derivatives ofGHRP-6, a study on the inhibition of vascular sprouting by DBG 178 aprototype of azapeptide derivatives at the range concentrations from10⁻⁸ to 10⁻⁶M were performed on Matrigel-embedded aortic rings collectedfrom CB57BL/6 mice and CD36 null mice on the same background. Sproutingaortic endothelium (Von Willebrand factor positive) expresses CD36 asdemonstrated by is immunochemistry (FIG. 1). FIG. 1 illustratesdisruption of vascular sprouting of aortic endothelium by azapeptidecompound DBG178. Sprouting aortic endothelium (Von Willebrand factorpositive) expressing CD36 is shown in the upper row of FIG. 1a .Vascular sprouting of aortic rings from C57BL/6 and CD36 null mice inthe presence of DBG 178 at 10⁻⁷M is shown in the lower row of FIG. 1a .Histograms of microvascular sprouts areas (mean±s.e.m) from aorticexplants of C57BL/6 and CD36 null mice exposed to DBG concentrationsfrom 10⁻⁸ to 10⁻⁶ M p<0.01 compared with control (Ctl) is shown in FIG.1 b.

DBG 178 significantly inhibited the vascular sprouting of aortic ringsfrom CB57BL/6 mice in a dose dependent manner as shown in FIG. 1. Incontrast this inhibitory effect on vascular sprouting of DBG178 was notdetected on aortic rings from CD36 null mice. DBG 178 appears to disruptvascular sprouting in vitro at concentrations ranging from 10⁻⁷ to 10⁻⁶M and this inhibitory effect is dependent of CD36 expression.

Aortic Rings Preparation and In Vitro Vascular Sprouting Protocol

Aortas were excised from 10-week-old male C57/Bl6+/+ and CD36−/− mice ofC57/Bl6 background. The fibroadipose tissue was removed and aortas weresectioned into 2-mm-long cross-sections. The +/+ and −/− rings werecovered by 50 μl of Matrigel and cultured for 4 days in EGM-2 medium(Clonetics Corp.). Explants were then exposed to vehicle alone or inpresence of an azapeptide prototype DBG178 at the indicatedconcentrations (10⁻⁸ to 10⁻⁶ M) from day 4 to day 6 of culture in EGM-2medium. Pictures of each individual explant were taken at day 4 and day6 and EC growth was measured as the surface covered at day 6 minus thesurface covered at day 4 Data were subject to analysis of variance(ANOVA) followed by Dunnet's post test to test for differences amonggroups. Outgrowing cells were characterized by double-labelling withmonoclonal vWF antibody (1:100, Dako) and polyclonal CD36 (1:100, SantaCruz) after acetone fixation using their respective alexa coupledsecondary antibodies.

Inhibition of Choroidal Neovascularisation In Vivo Using the LaserInjury Model

The standard model of choroidal neovascularisation is the laser injuryinduced neovascularisation (47). In this model, which is applicable torodents and primates, a laser beam is used to disrupt the RPE and theBruchs membrane that separates the choroidal vasculature from thesubretinal space. The following local inflammatory reaction in deepretina and choroid leads to a localized subretinal neovascularisation ina similar manner as that observed in AMD. This local neovascularisationcan be quantified on flatmounts of RPE/choroid/sclera. Theantiangiogenic effect of DBG178 on subretinal neovascularisation wastested by injecting intravitreally at effective concentration of 10⁻⁷ to10⁻⁶ M within the eye and compared to that of intravitreal injection ofsaline used as a control.

FIG. 2 illustrates choroidal vessels detected using FITC-conjugateddextran infusion following laser-induced posterior retinal injury ineyes from 10-week-old male C57BL/6 mice and CD36 deficient mice of thesame background with or without intravitreal treatment with DBG178 (FIG.2a ). Histograms of the surfaces of neovascular vessels obtained fromC57BL/6 (CD36_(+/+)) and CD36 null (CD36_(−/−)) mice with (DBG) orwithout (Ctl) treatment with DBG178 are illustrated in FIG. 2 b.

DBG 178 significantly inhibited the neovascular response by more than50% as shown in FIG. 2a . In contrast, DBG 178 had no effect on theneovascular response in the eye of CD36 null mice, showing that itsantiangiogenic effect is CD36 dependent. The CD36 null mice developedsignificantly less neovascularisation following laser injury as comparedto C57BL/6 mice which contrasted with an expected exaggeratedneovascularisation considering its role as the main TSP-1/2 receptor. Onthe other hand, interference with mechanisms of RPE phagocytosisexpected in CD36 deficiency could impede RPE expression of angiogenicmediators such as COX-2(50) leading to the diminishedneovascularisation.

Methodology for Laser Induced Choroidal Neovascularisation

Ten-week old C56/Bl6+/+ and CD36 null mice of C57Bl/6 background wereanesthetized by intramuscular injection of ketamine (50 mg/kg) andxylazine (10 mg/kg). Pupils were fully dilated with 1% tropicamide.Coverslips positioned on the mouse cornea were used as a contact glass.Laser-coagulations were performed 1 to 2 disc diameters away from thepapillae using an Argon laser (532 nm) mounted on a slit lamp (400 mW,50 ms and 50 μm). 3 laser impacts were applied to each eye. At day 3 andday 7 1 μl of azapeptide derivative DBG 178 in 0.9% NaCl at 10⁻⁶ M wasinjected into the vitreous using glass capillaries (ca. 60 gauge) and amicroinjector. Each treatment group contained a minimum of 4 mice (8eyes). At day 10 mice were perfused with Fluorescein Dextran at 10⁻⁶ M.Their eyes were enucleated, fixed in 4% PFA for 15 minutes at roomtemperature, sectioned at the limbus and the cornea and lens werediscarded. The retinas were carefully peeled from theRPE/choroid/sclera. Retinas and choroids were fixed for additional 15minutes in methanol at −20° C. and incubated with TRITC-conjugatedGriffonia simplicifolia Lectin (Sigma-Aldrich). The RPE/choroids wereradially incised, flatmounted and viewed using a fluorescence microscope(BX51; Olympus). CNV were measured on photographs using Scion imageanalysis software.

Table 3 below shows IC₅₀ values obtained from competition curves ofazapeptide derivatives in covalent photolabelling receptor assay usingrat cardiac membranes as source of CD36 and the photoactivatablehexarelin derivative [¹²⁵I]Tyr-Bpa-Ala-hexarelin as radioligand.

TABLE 3 Binding affinity of aza peptide derivatives towards CD36 Azapeptide/ Compound No. Structure IC₅₀ HexarelineHis-D-2-Me-Trp-Ala-Trp-D-Phe-Lys-NH₂ 2.97 × 10⁻⁶M DBG253-1His-D-Trp-Ala-azaPhe-D-Phe-Lys-NH₂ 1.34 × 10⁻⁶M DBG253-2His-D-Trp-Ala-azaLeu-D-Phe-Lys-NH₂ 2.89 × 10⁻³M DBG253-3His-D-Trp-Ala-azaBip-D-Phe-Lys-NH₂ 1.35 × 10⁻⁵M DBG253-4His-D-Trp-Ala-azaCha-D-Phe-Lys-NH₂ 2.10 × 10⁻³M DBG253-5His-D-Trp-Ala-azahomoPhe-D-Phe-Lys- >>10⁻⁵M NH₂ DBG253-6His-D-Trp-Ala-azaNa1-1-D-Phe-Lys-NH₂ 3.62 × 10⁻⁵M DBG253-7His-D-Trp-Ala-Tyr-D-Phe-Lys-NH₂ 1.32 × 10⁻⁵M DBG253-8His-D-Trp-Ala-D-Tyr-D-Phe-Lys-NH₂ 1.20 × 10⁻⁵M DBG253-9(D/L)-His-azaPhe-Ala-Trp-D-Phe-Lys- 7.24 × 10⁻⁵M NH₂ DBG253-10(D/L)-His-azaNa1-1-Ala-Trp-D-Phe-Lys- 1.93 × 10⁻³M NH₂ DBG253-11(D/L)-His-azahomoPhe-Ala-Trp-D-Phe- 3.68 × 10⁻⁵M Lys-NH₂ DBG253-12(D/L)-His-azaBip-Ala-Trp-D-Phe-Lys- 2.32 × 10⁻⁵M NH₂ DBG253-13(D/L)-His-azaCha-Ala-Trp-D-Phe-Lys- >>10⁻⁵M NH₂ DBG201-AHis-D-Trp-azaLeu-Trp-D-Phe-Lys-NH₂ 2.89 × 10⁻⁶M DBG175p(D/L)His-azaTyr-Ala-Trp-D-Phe-Lys-NH₂ 1.80 × 10⁻⁶M DBG178pHis-D-Trp-Ala-azaTyr-D-Phe-Lys-NH₂ 2.80 × 10⁻⁵M DBG188pHis-D-Trp-azaGly-Trp-D-Phe-Lys-NH₂ 9.61 × 10⁻⁶M

Table 4 below shows IC₅₀ values obtained from competition curves ofazapeptide derivatives of GHRP-6 in GHS-R1a radioreceptor assay usingLLCPK-1 membranes overexpressing GSH-R1a and radioiodinated (1-28) ratghrelin as tracer.

TABLE 4 Azapeptide EC50 DBG145 8.09 × 10⁻⁷M DBG168-8 5.14 × 10⁻⁷MDBG175p 8.53 × 10⁻⁶M DBG178p 1.57 × 10⁻⁵M DBG188p 8.08 × 10⁻⁷M DBG253-12.77 × 10⁻⁶M DBG253-2 1.95 × 10⁻⁵M DBG253-3 1.34 × 10⁻⁶M DBG253-4 4.28 ×10⁻⁶M DBG253-5 3.74 × 10⁻⁶M DBG253-6 7.23 × 10⁻⁷M DBG253-7 7.71 × 10⁻⁷MDBG253-8 3.25 × 10⁻⁶M DBG253-9 1.61 × 10⁻⁵M DBG253-10 4.65 × 10⁻⁷MDBG253-11 7.29 × 10⁻⁷ DBG253-12 1.64 × 10⁻⁶M DBG253-13 5.49 × 10⁻⁶MDBG201-A 1.20 × 10⁻⁶M Ghreline, rat 2.84 × 10⁻¹⁰M GHRP-6 3.65 × 10⁻⁹M

Tables 5 and 6 show the binding affinity of compound numbers 39-65towards GS-R1a and CD36. The reference standard is GHRP-6 for theghrelin receptor R1a and hexarelin which is the methylated form of GHRP6 with the methyl Trp residue in position 2. The methodology used forthe GHS-R1a and CD36 binding studies was as previously described.

TABLE 5 Entry/ IC₅₀ Binding Item No. Cpd No. Formula GHS-R1a GHRP-6His-D-Trp-Ala-Trp-D-Phe-Lys-NH₂ 6.08 × 10⁻⁹M HexarelineHis-D-2-Me-Trp-Ala-Trp-D-Phe-Lys-NH₂ 39 CP-1A (i) 5.10 × 10⁻⁵M 56 CP-2B(v) Ala-D-Trp-azaLeu-Trp-D-Phe-Lys-NH₂ 1.12 × 10⁻⁵M 41 CP-1A (iii)(D/L)His-AzaPhe-Ala-Ala-D-Phe-Lys-NH₂

 10⁻⁵M 42 CP-1A (iv) Ala-AzaPhe-Ala-Trp-D-Phe-Lys-NH₂

 10⁻⁵M 43 CP-1B (i) His-AzaTyr-Ala-Trp-D-Phe-Ala-NH₂

 10⁻⁵M 44 CP-1B (ii) His-AzaTyr-Ala-Trp-D-Ala-Lys-NH₂

 10⁻⁵M 46 CP-1B (iv) Ala-AzaTyr-Ala-Trp-D-Phe-Lys-NH₂

 10⁻⁵M 53 CP-2B (ii) His-D-Trp-azaLeu-Trp-D-Ala-Lys-NH₂

 10⁻⁵M 54 CP-2B (iii) His-D-Trp-AzaLeu-Ala-D-Phe-Lys-NH₂

 10⁻⁵M 52 CP-2B (i) His-D-Trp-AzaLeu-Trp-D-Phe-Ala-NH₂ 8.17 × 10⁻⁶M 55CP-2B (iv) His-D-Ala-AzaLeu-Trp-D-Phe-Lys-NH₂

 10⁻⁵M 56 CP-2B (v) Ala-D-Trp-AzaLeu-Trp-D-Phe-Lys-NH₂

 10⁻⁵M 59 CP-3 (iii) His-D-Ala-Ala-AzaPhe-D-Phe-Lys-NH₂

 10⁻⁵M 60 CP-3 (iv) Ala-D-Trp-Ala-AzaPhe-D-Phe-Lys-NH₂

 10⁻⁵M 61 CP-AzaGly-Pro His-D-Trp-AzaGly-Pro-D-Phe-Lys-NH₂

 10⁻⁵M 62 CP-AP_4 His-D-Trp-AzaPro-Trp-D-Ala-Lys-NH₂

 10⁻⁵M 39 CP-1A(i) His-AzaPhe-Ala-Trp-D-Phe-Ala-NH₂

 10⁻⁵M 40 CP-1A(ii) His-AzaPhe-Ala-Trp-D-Ala Lys-NH₂

 10⁻⁵M 45 CP-1B(iii) His-AzaTyr-Ala-Ala-D-Phe-Lys-NH₂

 10⁻⁵M 47 CP-2A(i) His-D-Trp-AzaGly-Trp-D-Phe-Ala-NH₂ 4.50 × 10⁻⁵M 48CP-2A(ii) His-D-Trp-AzaGly-Trp-D-Ala-Lys-NH₂

 10⁻⁵M 49 CP-2A(iii) His-D-Trp-AzaGly-Ala-D-Phe-Lys-NH₂

 10⁻⁵M 50 CP-2A(iv) His-D-Ala-AzaGly-Trp-D-Phe-Lys-NH₂

 10⁻⁵M 51 CP-2A(v) His-D-Trp-AzaGly-Trp-D-Phe-Lys-NH₂ 5.68 × 10⁻⁷M 57CP-3(i) His-D-Trp-Ala-AzaPhe-D-Phe-Ala-NH₂

 10⁻⁵M 58 CP-3(ii) His-D-Trp-Ala-AzaPhe-D-Ala-Lys-NH₂

 10⁻⁵M 64 CP-AP-2 His-D-Trp-Ala-Trp-AzaPro-Lys-NH₂

 10⁻⁵M 63 CP-AP-3 His-D-Trp-Ala-AzaPro-D-Phe-Lys-NH₂

 10⁻⁵M 65 CP-AP-5 His-AzaPro-Ala-Trp-D-Phe-Lys-NH₂

 10⁻⁵M 66 ZS554-F29 Phe-D-Trp-Ala-AzaTyr-D-Phe-Lys-NH₂

 10⁻⁵M 67 ZS555-F40 Ala-D-Trp-Ala-AzaTyr-D-Phe-Lys-NH₂

 10⁻⁵M 68 ZS556 Hydrocinnamic acid-D-Trp-Ala-AzaTyr-D-Phe-Lys-

 10⁻⁵M NH₂

TABLE 6 Item No./ IC₅₀ Binding Entry No. Cpd. No. Formula CD36 GHRP-6His-D-Trp-Ala-Trp-D-Phe-Lys-NH₂ HexarelineHis-D-2-Me-Trp-Ala-Trp-D-Phe-Lys-NH₂ 3.33 × 10⁻⁶M 39 CP-1A (i)

 10⁻⁵M 56 CP-2B (v) Ala-D-Trp-azaLeu-Trp-D-Phe-Lys-NH₂ 3.68 × 10⁻⁶M 41CP-1A (iii) (D/L)His-AzaPhe-Ala-Ala-D-Phe-Lys-NH₂ 2.76 × 10⁻⁵M 42 CP-1A(iv) Ala-AzaPhe-Ala-Trp-D-Phe-Lys-NH₂ 5.02 × 10⁻⁶M 43 CP-1B (i)His-AzaTyr-Ala-Trp-D-Phe-Ala-NH₂ 7.22 × 10⁻⁵M 44 CP-1B (ii)His-AzaTyr-Ala-Trp-D-Ala-Lys-NH₂

 10⁻⁵M 46 CP-1B (iv) Ala-AzaTyr-Ala-Trp-D-Phe-Lys-NH₂ 1.74 × 10⁻⁵M 53CP-2B (ii) His-D-Trp AzaLeu-Trp-D-Ala-Lys-NH₂ 4.94 × 10⁻⁶M 54 CP-2B(iii) His-D-Trp-AzaLeu-Ala-D-Phe-Lys-NH₂ 9.66 × 10⁻⁶M 52 CP-2B (i)His-D-Trp-AzaLeu-Trp-D-Phe-Ala-NH₂ 9.02 × 10⁻⁶M 51 CP-2A(v)His-D-Trp-AzaGly-Trp-D-Phe-Lys-NH₂ 8.76 × 10⁻⁶M 64 CP-AP-2His-D-Trp-Ala-Trp-AzaPro-Lys-NH₂

 10⁻⁵M 63 CP-AP-3 His-D-Trp-Ala-AzaPro-D-Phe-Lys-NH₂

 10⁻⁵M 66 ZS554-F29 Phe-D-Trp-Ala-AzaTyr-D-Phe-Lys-NH₂ 1.18 × 10⁻⁵M 67ZS555-F40 Ala-D-Trp-Ala-AzaTyr-D-Phe-Lys-NH₂ 2.95 × 10⁻⁶M 68 ZS556Hydrocinnamyl-D-Trp-Ala-AzaTyr-D-Phe-Lys-NH₂ 3.25 × 10⁻⁵M

For azapeptides CP-2B(v) and CP-1A(iv), the modification of thestructure of GHRP-6 decreased drastically the binding affinity towardsR1a receptor without major change in the affinity to the CD36 receptorcompared with hexarelin, the methylated derivative of GHRP6. The samewas observed for CP-2B(ii) and ZS555-F40.

Alternative Azapeptides Synthesis Method

The present invention also extends to an alternative method ofsynthesizing azapeptides, as described below. A variety of methods havebeen developed for the introduction of aza-amino acids into peptides bysolution-phase synthesis (49). Few strategies have, however, beenreported for preparing aza-peptides on solid support and only recentlyhave azapeptide libraries been synthesized. In preliminary attempts tomake aza-peptides, the N-terminal amino group of a resin bound peptidewas converted into an isocyanate or active carbamate and subsequentlyreacted with a suitable N′-alkyl fluoren-9-yl methyl carbamate. Thisstrategy afforded the desired aza-peptide contaminated with significantamounts of hydantoin (50) and to avoid the latter, additional backboneamide protection was required (50b). Solution-made aza-tri or dipeptidefragments have been coupled to the N-terminus of resin-bound peptides(40, 51). Multiple synthetic steps were required to makeN-(Boc)aza-dipeptides, that have served as configurationally stablebuilding blocks for Boc/Bzl solid-phase peptide synthesis (40). In thefirst effective Fmoc-strategy for solid-phase aza-peptide synthesis, thereaction of N-Fmoc protected aza-amino acid chlorides onto the growingchain of a resin-bound peptide has provided access to a variety ofaza-peptide analogs (39). This procedure consists of solution-phasesynthesis of N-substituted fluorenylmethyl carbazates by reductiveamination, followed by activation to the N-Fmoc-aza-amino acid chlorideand coupling to the resin bound peptide.

Aza-amino scanning would constitute an effective means for identifyingthe importance of turn structure for activity in native peptides. Theuse of Fmoc-aza-amino acid chlorides has realized itself as a moregeneral and efficient procedure for the solid-phase preparation ofaza-peptides and in determining their structure-activity relationshipsagainst biological targets (41). However, this methodology has not beenwithout its limitations; in particular, the scope of N′-substitutedfluorenylmethyl carbazates is constrained by the prerequisite of asolution-phase synthesis of the N-substituted carbazates. The narrowrange of functionalized aldehydes that may be amenable to reductiveamination procedures has created a desire for alternative processes foradding the side-chain onto the aza-amino acid residue.

Currently, most synthetic routes to modify peptides incorporate theindividually prepared un-natural amino acid residue by conventionalsolid phase peptide synthesis (SPPS) (52). A more direct and flexiblemethod for the preparation of peptidomimetics would be to build the newside-chains onto the growing peptide strand during the peptide synthesis(53). Aza-peptides possess one or more aza-amino acid residues in whichthe α-carbon is substituted for a nitrogen atom. In contrast to theenantioselective synthesis of natural amino acid residues, thecarbon-nitrogen bond forming reaction for aza-amino acid residues doesnot require stereo-chemical control. Selective introduction of theside-chain at the α-nitrogen of an aza-peptide does demandchemo-selective alkylation and chain extension reactions at anaza-glycine residue in the growing peptide chain on resin. In thepresent novel approach, activated aza-glycine residues are incorporatedinto the peptide chain by treating arylhydrazones with a phosgeneequivalent such as p-nitrophenyl chloroformate and acylation ofsupport-bound peptides and amino acids to form the respective N-terminalsemicarbazones. Alkylation of the semicarbazone may then be achievedregio-selectively to introduce varying side-chain groups. Thisregio-selective carbon-nitrogen bond forming sequence for the synthesisof structurally diverse aza-peptides followed a three-step procedureinserted into a conventional solid-phase peptide synthesis (SPPS) cycle:(FIG. 5, reactions in box), activation (A), alkylation (B), andsemicarbazone deprotection (C), followed by the normal SPPS sequenceinvolving coupling (D), deprotection and cleavage from the support (E).Following step C in the loop a number of options are possible, includingrepeating the steps in the synthesis cycle to add another unnaturalresidue to the growing peptide chain, coupling a natural amino acid orother residue via normal solid-phase methodology, or protecting the freeamino group subsequent to other reactions on the resin-bound product.

The present analysis was initiated with conventional SPPS of a knowndipeptide [D-Phe-Lys] and tripeptide [Trp-D-Phe-Lys], which constitutethe start sequence of a member of the growth hormone releasing peptidefamily namely, GHRP-6 (His-D-Trp-Ala-Trp-D-Phe-Lys-NH2). Thishexapeptide acts at the growth hormone secretagogue and stimulates therelease of growth hormones from the pituitary (54, 55). Syntheses wereconducted on a hydrophilic NovaPEG Rink Amide Resin™ which displayedexcellent swelling properties in polar protic solvents, including waterand MeOH (56).

Phenyl hydrazone was prepared on condensation of benzaldehyde andhydrazine hydrate. Treatment of phenyl hydrazone with p-nitrophenylchloroformate (57), provides the imine protected activated aza-glycineintermediate for coupling to the N-terminus of the peptide bound resin.Other reagents such as phosgene (39) and carbonyldiimidazole (58), whichhave previously been used in solution for the synthesis of aza-peptides,instead, gave the symmetric urea from reaction with two equivalents ofhydrazone. The acylation of the resin-bound peptide was efficientlymonitored by LCMS analysis after deprotection and cleavage of peptidefrom a small aliquot (3 mg) of resin with a freshly made TFA/TES/H₂O(95:2.5:2.5, v/v/v) solution. This indicated complete conversion of thestarting material to the resin-bound semicarbazone peptide 2i (FIG. 4)

In analogy to the carbon-nitrogen double bond resonance stabilizing thealpha-carbanion of a glycine Shiff-base in the synthesis ofα-substituted amino acid analogs (59), the semicarbazone was expected tofavor deprotonation and alkylation of aza-glycine 2i. In semicarbazone2i, the proton on the hydrazone nitrogen is made sufficiently acidicsuch that basic conditions may be used to effect regio-selectivedeprotonation for alkylation reactions (60). The regio-selectivedeprotonation and alkylation reactions were first tried in-solutionusing the benzophenone semicarbazone, aza-glycinyl-glycine ethyl ester5i, which was derived from activation of the hydrazone from bezophenoneusing p-nitrophenyl chloroformate and acylation of glycine ethyl ester(FIG. 5). Semicarbazone 5i (100 mol %) was treated with potassiumt-butoxide (120 mol %) as base and allyl bromide (120 mol %) aselectrophile to yield 60% of a single allylated product, benzophenonesemicarbazone aza-(allyl)Gly-Gly-OEt 6i, for which the ¹H—¹H COSY NMRspectrum indicated a coupling cross-peak between the amide NH and theglycine methylene protons, as evidence of the regio-selectivemono-alkylation reaction.

On solid support, benzaldehyde semicarbazone 2i proved efficient inalkylations using potassium t-butoxide (300 mol %) and variouselectrophiles (300 mol %) to give monoalkylated products 7ai-7gi (FIG.6) as characterized by their analytical LCMS traces after cleavage of asmall aliquot of peptide from the resin. In the case of 7ai and 7di, thehydrochlorides required an additional equivalent of base in the reactionmixture to effect neutralization of the salt. In the case of product7bi, the alkylation reaction was found to be sluggish with potassiumt-butoxide as base favoring only 20% conversion. However, the strongerorganic soluble non-ionic phosphazene (Schwesinger) base (61),tert-butylimino-(BTPP) gave 85% conversion to the alkylated product 7bi.In the case of the more sterically encumbered secondary alkyl halides,with postassium f-butoxide as base, isopropyl bromide failed to react;however, alkylation proceeded cleanly with isopropyl iodide, resultingin complete conversion to the aza-valine analog 7fi.

Hydrazones have been used as chiral auxiliaries in organic processesrelated to the enantioselective carbonyl group transformations (62).They are easily introduced, chemically robust and tolerant to a highdegree of functionality. Their removal has, however, necessitated use ofharsh conditions, i.e., strong acid, oxidative and reductive cleavageconditions to release the hydrazide moiety, sometimes in a modified form(63). Some of the milder deprotection conditions associated with acidcatalyzed hydrolysis (64) and Lewis-acid promoted dithioketalization(65) favor the release of the free hydrazide moiety. Since Fmoc SPPSmethodology is regarded as an acid sensitive strategy, due to theemployment of the acid-labile Rink amide linker and side-chainprotecting groups, milder methods were developed for the incorporationand removal of the semicarbazone group under essentially neutralconditions. Preliminary trials were conducted on a Rink amide linkerpolystyrene based resin. Acid catalyzed hydrolysis reactions andLewis-acid promoted dithioketalization deprotection, both resulted inpartial to complete release of the semicarbazone with concomitantremoval of side-chain protecting groups from the peptide-bound resin andformation of side-products. Conversely, a mild hydrolysis procedure hasbeen described for the removal of imine-protecting groups from thepeptide bound resin (53), employing aqueous NH₂OH—HCl, THF, pH=6,followed by free-basing with diisopropylethylamine (DIPEA). In ourhands, no solvolysis of semicarbazone 7i was observed to occur underthese identical conditions on a hydrophobic polystyrene based resin;however, when the more hydrophilic NovaPEG Rink Amide Resin™ wasemployed, the resin swelled in the hydrolytic solvent conditionsfavoring deprotection (66). Complete liberation of semicarbazides8ai-8gi, from semicarbazones 7ai-7gi, was accomplished using elevatedtemperatures (60° C., FIG. 7) as determined by their analytical LCMStraces obtained after cleavage of a small aliquot of peptide from theresin. The reaction also produced benzaldehyde oxime (9i) as by-product,indicating that the reaction occurred by imine exchange and nothydrolysis.

Acylation of the aza-amino acid residue was performed as described inthe literature (51), by treatment of the resin swollen in THF with a6-fold excess of Fmoc-amino acid chloride, generated in situ withbis-(trichloromethyl)-carbonate (BTC) (67) and 2,4,6-collidine.Aza-peptides were analyzed by LCMS, which indicated a crude-purityranging from 40-60% of the desired acylated products. Aza-peptidesyntheses were then continued by the conventional SPPS (68). Finalcleavage of aza-peptides from the support and deprotection of side-chainprotecting groups were performed by treating the resin with a freshlymade TFA/TES/H₂O (95:2.5:2.5, v/v/v) solution. Aza-peptides (Table 6)were isolated after evaporation, dissolved in a 1:1 acetonitrile/H₂Osolution, and lyophilized to white foams that were directly analyzed byLCMS to assess a crude purity ranging from 8-20%. These weresubsequently purified to determine the pure yield recovery (FIG. 8).

TABLE 7 Yields and Purities of isolated Azapeptides. T_(R) (min) T_(R)(min) HPLC in in purity at Yield Expected Compound ACN^(a) MEOH 214nm^(b) (%)^(c) Mass Mass^(d) 10ai 13.76 17.38 94.8% 6.53 913.86 913.5,916.2^(e) 10bi 10.03 n/a 36.2% 2.62 835.95 836.4 10ci 10.84 13.86 92.8%3.44 874 874.4 10di 9.45 10.82 95.4% 3.33 898.02 900.4 ^(a)Unlessotherwise noted, analytical HPLC analyses were performed on a 5 μM 150mm × 4.6 mm C18 Gemini column with a flow rate of 0.5 ml/min using a0-40 gradient from water (0.1% FA) to CH₃CN (0.1% FA) or MeOH (0.1% FA).^(b)HPLC purity at 214 nm of the purified peptide. ^(c)Yields afterpurification by HPLC are based on manufacturer's reported loading forRink resin. ^(d)Observed masses corresponding to the H⁺ adducts.^(e)Presence of Br yields 50:50 ratio of M⁺ and M + 2 isotopes.

These preliminary studies demonstrate that the submonomer approach canbe utilized to convert resin-bound amino acids or peptides to unnaturalaza-peptide derivatives. The mild reagents and conditions used arecompatible with Fmoc based SPPS strategies. A more structurally diversegroup of novel aza-peptides are now accessible using this technique.Therefore, this method can be readily applicable to aza-amino scanningand identifying the importance of turn structure for activity in nativepeptides and in determining their structure-activity relationshipsagainst biological targets. Furthermore this method may prove effectivefor synthesizing libraries of aza-peptides by combinatorial methods suchas IRORI kan technology.

General Methods. NovaPEG™ Rink Amide resin (0.67 mmol/g) was purchasedfrom NovaBiochem® Inc., and the manufacturer's reported loading of theresin was used in the calculation of the yields of the final products.Reagents such as benzaldehyde, p-nitrobenzyl chloroformate, potassiumt-butoxide, tert-butylimino-tri(pyrrolidino)phosphorane (BTPP),2-(chloromethyl)quinoline hydrochloride, 4-bromobenzyl bromide,2-(chloromethyl)pyridine hydrochloride, iodomethane, 2-iodopropane, 80%propargyl bromide in toluene and hydroxylamine hydrochloride werepurchased from Aldrich and used directly. The alkylating reagent,3-(2-bromoethyl) indole was also purchased from Aldrich, but protectedas the N-Boc analogue as described in the literature (69) prior to thealkylation reaction. ¹H and ¹³C NMR spectra were recorded, respectively,at 400 and 100 MHz in CDCl₃ as the solvent and tetramethylsilaneinternal reference. Thin-layer chromatography was performed on silicagel 60 F254 plates from Merck™. Flash chromatography was performed onsilica gel 60 (230-400 Mesh ASTM) from Merck™. Commercially availableFmoc amino acids and HBTU were purchased from GL Biochem™ and used asreceived. Solvents for reactions were dried and distilled prior to use.Analytical LCMS and HPLC analyses were performed on either a 5 μM, 150or 50 mm×4.6 mm C18 Phenomenex Gemini Column™ with a flow rate of 0.5ml/min using either a 20-80% or a 0-40% gradient from water (0.1% FA) toCH₃CN (0.1% FA) or MeOH (0.1% FA). Aza-peptides 10ai-di were purified ona semi-preparative column (5 μM, 250 mm×21.2 mm, C18 Gemini Column™)using a 2-40% gradient from water (0.1% FA) to CH₃CN (0.1% FA) with aflow rate of 10.6 ml/min.

Benzophenone semicarbazone aza-glycinyl-glycine ethyl ester, 5i

Benzophenone hydrazone (500 mg, 2.5 mmol) in DCM (5 mL) was addeddropwise over 15 min to a solution of p-nitrobenzyl chloroformate (600mg, 3 mmol) in DCM (5 mL) at 0° C. The reaction mixture was stirred atroom temperature under argon for an additional 1.5 h, until TLC, [(2:1Hex:EtOAc), Rf (benzophenone hydrazone): 0.45 and Rf (3i): 0.7]indicated complete consumption of the starting material. To this mixturewas added dropwise DIPEA (400 μL, 4.6 mmol) and the suspension wasstirred for an additional 3 h at room temperature, (22° C.) under argon.The reaction mixture was diluted in DCM (60 mL) and extracted severaltimes with NaHCO₃ (8×30 mL). The organic phase was dried over MgSO₄,concentrated in-vacuo and purified by flash chromatography using 2:1Hexane:EtOAc [Rf (5i): 0.25] to yield a white solid (0.5 g, 60%), ¹H NMR(CDCl₃) δ: 1.19 (t, J=7 Hz, 3H), 4.02 (d, J=5.6 Hz, 2H), 4.13 (q, J=7Hz, 2H), 6.78 (t, J=5.5 Hz, 1H), 7.1-7.4 (m, 10H), 7.61 (s, 1H); ¹³C NMR(CDCl₃) δ: 13.8, 41.4, 61.0, 126.8, 127.8, 127.9, 128.1, 129.0, 129.1,129.36, 129.43, 129.5, 131.4, 136.5, 148.2, 151.0, 154.9, 170.0; LCMS(ESI) Calcd. for C₁₈H₁₉N₃O₃, M⁺: 325.4. found (M+H)⁺ 326.

Benzophenone semicarbazone aza-allylglycinyl-glycine ethyl ester, 6i

To a solution of semicarbazone 5i, (50 mg, 0.15 mmol) in THF (0.5 mL),potassium t-butoxide (25 mg, 0.18 mmol) was added at 0° C. under argon.The reaction mixture was stirred for 1 h, treated drop-wise with allylbromide (20 μL, 0.18 mmol), stirred for 3 h at room temperature (22° C.)under argon, diluted in Et₂O (5 mL) and extracted with NaHCO₃ (2×2 mL)and brine (2×2 mL). The organic phase was dried over MgSO₄, concentratedin-vacuo and purified by flash chromatography using 2:1 Hexane:EtOAc [Rf(6i): 0.45] to yield a an oil (35 mg, 60%). ¹H NMR (CDCl₃) δ: 1.23 (t,J=7 Hz, 3H), 3.86 (dd, J=1.6 Hz, 3.8 Hz, 2H), 4.05 (d, J=5.7 Hz, 2H),4.16 (q, J=7 Hz, 2H), 4.78 (dd, J=1 Hz, 10 Hz, 1H), 4.92 (dd, J=1 Hz, 10Hz, 1H) 5.33-5.43 (m, 1H), 6.89 (t, J=5.5 Hz, 1H), 7.18-7.42 (m, 10H);¹³C NMR (CDCl₃) δ: 13.8, 42.2, 47.9, 60.8, 116.3, 127.8, 128.2, 128.3,128.7, 129.3, 129.6, 132.4, 135.6, 138.4, 157.7, 158.4, 170.2; LCMS(ESI) Calcd. for C₂₁H₂₃N₃O₃, M⁺: 365.4. found (M+H)⁺ 366.1.

General Procedure for Peptide Synthesis, Fmoc Deprotection and HBTUCouplings

Peptide syntheses were performed under standard conditions (70) on anautomated shaker using NovaPEG™ Rink Amide resin (0.67 mmol/g).Couplings of amino acids (3 equiv) were performed in DMF using HBTU (3equiv) as coupling reagent and DIPEA (6 equiv). Fmoc deprotections wereperformed by treating the resin with 20% piperidine in DMF for periodsof 10 and 20 min. Resin was washed after each coupling and deprotectionstep sequentially with DMF (2×10 mL), MeOH (2×10 mL), EtOH (2×10 mL),THF (2×10 mL), Et₂O (2×10 mL) and DCM (2×10 mL). The coupling steps weremonitored to completion by the Kaiser (70) test.

General Procedure for the Preparation of Benzladehyde SemicarbazonePeptide Resin 2i

To a stirred solution of EtOH (1.5 mL) and hydrazine hydrate (60 μL, 1.8mmol) at 0° C., benzaldehyde (60 μL, 0.6 mmol) was added dropwise togenerate the phenyl hydrazone. The reaction was stirred to completionwhich was usually after 15 min as indicated by TLC, [(2:1 Hex:EtOAc), Rf(benzaldehyde): 0.7 and Rf (benzaldehyde hydrazone): 0.6], and poureddirectly into H₂O (5 mL) and extracted in DCM (3×5 mL). The organicphase was dried with MgSO₄ and concentrated in-vacuo to yield thebenzaldehyde hydrazone as a yellow-tinged oil that was employed directlywithout further purification.

Benzaldehyde hydrazone (70 mg, 0.6 mmol, 3 equiv.) in DCM (1 mL) wasadded dropwise over 15 min to a solution of p-nitrobenzyl chloroformate(125 mg, 0.61 mmol, 3.2 equiv.) in DOM (1 mL) at 0° C. The reactionmixture was stirred at room temperature under argon for an additional1.5 h, until TLC, [(2:1 Hexane:EtOAc), Rf (benzaldehyde hydrazone); 0.6and Rf (1i): 0.75] indicated complete consumption of the startingmaterial. To this mixture was added dropwise DIPEA (210 μL, 1.2 mmol, 6equiv.) and the suspension was quickly transferred to the resin. Thereaction was mixed on an automated shaker for 6 h at room temperature.The resin was filtered and washed under vacuum with DMF (2×10 mL), MeOH(2×10 mL), EtOH (2×10 mL), THF (2×10 mL), Et₂O (2×10 mL) and DCM (2×10mL). The extent of reaction conversion was monitored on an aliquot (3mg) of resin which was subjected to 1 mL of TFA/TES/H₂O (95:2.5:2.5,v/v/v) for resin cleavage and the crude was analyzed by LCMS.

Benzaldehyde semicarbazone Aza-Gly-D-Phe-Lys-NH₂ (2ai)

Tr=3.77 min; LCMS (ESI) calcd for C₂₃H₃₀N₆O₃ (M+2H)⁺, 440.6. found m/e441.3 (M+2H)⁺.

Benzaldehyde semicarbazone Aza-Gly-Trp-D-Phe-Lys-NH₂ (2bi). Tr=4.38 min;LCMS (ESI) calcd for C₂₇H₃₆N₈O₄ (M+2H)⁺, 626.7. found m/e 627.4 (M+2H)⁺.

General Procedure for the Synthesis of Mono-Alkylated BenzaldehydeSemicarbazone Peptide Resin 7i

To the swollen benzaldehyde semicarbazone peptide bound resin 2i (0.1 g,67 μmol) in THF (2 mL), potassium t-butoxide (25 mg, 0.2 mmol, 3 equiv.)or BTPP (for the synthesis of 7bi, 60 μL, 0.2 mmol, 3 equiv.) was added.The mixture was agitated on an automated shaker for 1 h, treated withthe electrophile, R—X (0.2 mmol, 3 equiv.) and agitated on the shaker atroom temperature for an additional 12 h. The resin was filtered andwashed under vacuum with DMF (2×10 mL), MeOH (2×10 mL), EtOH (2×10 mL),THF (2×10 mL), Et₂O (2×10 mL) and DCM (2×10 mL). The extent of reactionwas monitored by subjecting an aliquot (3 mg) of resin to cleavage [1mL, TFA/TES/H₂O (95:2.5:2.5, v/v/v)] and analyzing the crude by LCMS.

Benzaldehyde semicarbazone Aza-(2-quinolinyl)Ala-D-Phe-Lys-NH₂ (7ai).Tr=4.59 min; LCMS (ESI) calcd for C₃₃H₃₇N₇O₃ (M+H)⁺, 579.7. found m/e580.3 (M+H)⁺.

Benzaldehyde semicarbazone Aza-(N-Boc-3-ethylindolyl)Gly-D-Phe-Lys-NH₂(7bi). Tr=4.52 min; LCMS (ESI) calcd for C₃₃H₃₉N₇O₃ (M+2H)⁺, 581.8.found m/e 584.4 (M+2H)⁺.

Benzaldehyde semicarbazone Aza-(p-bromo)Phe-D-Phe-Lys-NH₂ (7ci). Tr=4.70min; LCMS (ESI) calcd for C₃₀H₃₅BrN₆O₃ (M+H)⁺, 607.5. found m/e 609.3and 611.2 (M+H)⁺ in a 1:1 ratio due to Br isotopes.

Benzaldehyde semicarbazone-Aza-(2-pyridyl)Ala-D-Phe-Lys-NH₂ (7di).Tr=3.69 min; LCMS (ESI) calcd for C₂₉H₃₅N₇O₃ (M+H)⁺, 530.6. found m/e530.3 (M+H)⁺.

Benzaldehyde semicarbazone-Aza-Ala-Trp-D-Phe-Lys-NH₂ (7ei). Tr=4.56 min;LCMS (ESI) calcd for C₃₅H₄₂N₈O₄ (M+2H)⁺, 640.9. found m/e 641.4 (M+2H)⁺.

Benzaldehyde semicarbazone Aza-Val-Trp-D-Phe-Lys-NH₂ (7fi). Tr=4.58 min;LCMS (ESI) calcd for C₃₇H₄₆N₈O₄ (M+2H)⁺, 668.9. found m/e 669.4 (M+2H)⁺.

Benzaldehyde semicarbazone Aza-(propargyl)Gly-Trp-D-Phe-Lys-NH₂ (7gi).Tr=4.51 min; LCMS (ESI) calcd for C₃₇H₄₂N₈O₄ (M+2H)⁺, 664.9. found m/e665.4 (M+2H)⁺.

General Procedure for the Deprotection of the Benzaldehyde SemicarbazonePeptide Resin 7i Synthesis of Semicarbazido Peptide Resin 8Ai-8Gi

Resin-bound semicarbazone 7ai-7gi (0.1 g, 67 μmol) was washed with MeOHand then MeOH:H₂O (2:1, 3×1.5-2 mL). A solution of 1.6 M NH₂OH—HCl inMeOH:H₂O (2:1 v/v) (1 mL) was added to the resin and the suspension washeated in an oil-bath at 60° C. for 24 h. The resin was filtered andwashed under vacuum with DMF (2×10 mL), MeOH (2×10 mL), EtOH (2×10 mL),THF (2×10 mL), Et₂O (2×10 mL) and DCM (2×10 mL), and then neutralizedwith 50% DIEA:THF (2 mL) for 1 h. The resin was drained and washed asbefore and the extent of reaction was monitored by subjecting an aliquot(3 mg) of resin to the cleavage conditions [1 mL, TFA/TES/H₂O(95:2.5:2.5, v/v/v)] and analyzing the crude by LCMS.

Aza-(2-quinolinyl)Ala-D-Phe-Lys-NH₂ (8ai). Tr=2.89 min; LCMS (ESI) calcdfor C₂₆H₃₃N₇O₃ M⁺, 491.6 and (M+Na)⁺514.6. found m/e 492.3 M⁺ and(M+Na)⁺514.4.

Aza-(3-ethylindolyl)Gly-D-Phe-Lys-NH₂ (8bi). Tr=3.99 and 2.82 min; LCMS(ESI) calcd for C₂₆H₃₅N₇O₃M⁺, 493.6. found m/e 494.3 M⁺ and 351.2(M-3-ethylindole)⁺.

Aza-(p-bromo)Phe-D-Phe-Lys-NH₂ (8ci). Tr=4.17 min; LCMS (ESI) calcd forC₂₃H₃₁BrN₆O₃M⁺, 519.4. found m/e 519.3 and 521.3, M⁺ in a 1:1 ratio dueto Br isotopes.

Aza-(2-pyridyl)Ala-D-Phe-Lys-NH₂ (8di). Tr=2.88 min; LCMS (ESI) calcdfor C₂₂H₃₁N₇O₃M⁺, 441.5. found m/e 442.3 M⁺.

Aza-Ala-Trp-D-Phe-Lys-NH₂ (8ei). Tr=3.76 min; LCMS (ESI) calcd forC₂₈H₃₈N₈O₄ M⁺, 550.6. found m/e 551.4 M⁺.

Aza-Val-Trp-D-Phe-Lys-NH₂ (8fi). Tr=4.10 min; LCMS (ESI) calcd forC₃₀H₄₂N₈O₄M⁺, 578.7. found m/e 579:4 (M+2H)⁺.

Aza-(propargyl)Gly-Trp-D-Phe-Lys-NH₂ (8gi). Tr=3.95 min; LCMS (ESI)calcd for C₃₀H₃₈N₈O₄M⁺, 574.7. found m/e 575.4 M⁺.

General Procedure for Coupling of the Next Amino Acid to theSemicarbazido Peptide Resin 8i

Semicarbazido peptide resin 8ai-8gi (0.1 g, 67 μmol) was swollen in dryTHF (1 mL) and treated with a solution of Fmoc-amino acid (0.4 mmol, 6equiv.) in THF (1 mL), followed sequentially by BTC (0.2 mmol, 3 equiv)and 2,4,6-collidine (2 mmol, 30 equiv.). The resin was filtered andwashed under vacuum with DMF (2×10 mL), MeOH (2×10 mL), EtOH (2×10 mL),THF (2×10 mL), Et₂O (2×10 mL) and DCM (2×10 mL). The extent of reactionwas monitored by subjecting an aliquot (3 mg) of resin to the cleavageconditions [1 mL, TFA/TES/H₂O (95:2.5:2.5, v/v/v)] and analyzing thecrude by LCMS. The target sequences were completed according to theconventional SPPS.

General Procedure for Side Chain Deprotection and Aza-Peptide Cleavage

Aza-peptide resin was treated with a freshly made solution ofTFA/H₂O/TES (95:2.5:2.5, v/v/v, 20 mL/g of aza-peptide resin) for 2 h atroom temperature. The cleavage mixture was filtered, and the resin waswashed with neat TFA. The filtrate was then concentrated and dissolvedin an acetonitrile/H₂O (1:1, v/v) solution and lyophilized to yield alight foam or powder. The aza-peptides 10ai-di were purified bypreparative RP-HPLC using a semi-preparative column as described in thegeneral section.

His-D-Trp-Ala-aza-(p-bromo)Phe-D-Phe-Lys-NH₂ (entry 69 in Table, 10ai inFIG. 8). Tr₁=13.76 min; Tr₂=17.38 min; LCMS (ESI) calcd forC₄₃H₅₃BrN₁₂O₆ M⁺, 913.9. found m/e 913.5 and 916.2, M⁺ in a 1:1 ratiodue to Br isotopes.

His-D-Trp-Ala-aza-(2-pyridyl)Ala-D-Phe-Lys-NH₂ (10bi). Tr=10.03 min;LCMS (ESI) calcd for C₄₂H₅₃N₁₃O₆M⁺, 836.4 found m/e 442.3 M⁺.

His-D-Trp-aza-Ala-Trp-D-Phe-Lys-NH₂ (entry 12 in Table, 10ci in FIG. 8).Tr₁=10.84 min; Tr₂=13.86 min; LCMS (ESI) calcd for C₄₅H₅₅N₁₃O₆ M⁺, 874.found m/e 874.4 M⁺.

His-D-Trp-aza-(propargyl)Gly-Trp-D-Phe-Lys-NH₂ (entry 70 in Table, 10disuch as beta-alanine, beta-homophenylalanine as well as longer chainamino acids such)

Tr₁=9.45 min; Tr₂=10.82 min; LCMS (ESI) calcd for C₄₇H₅₄N₁₃O₆ (M+2)⁺,900. found m/e 900.4 (M+2)⁺.

REFERENCES

-   1. Klein R. Age related macular degeneration. Berger J W, Fine S L,    and Macguir M G ed, 1999; 17-30, Mosby, st louis, Missouri.-   2. De Jong P. Age related macular degeneration. New Engl. J. Med,    2006; 355:1474-1485,-   3. Rattner A, Nathans J. Macular degeneration: recent advances and    therapeutic opportunities. Nature Reviews Neuroscienc. 2006;    7:860-872-   4. Sarks S H. Ageing and degeneration in the macular region: a    clinico-pathological study. Brit J opthalmol 1976; 60:324-341-   5. Friedman E. The role of the atherosclerotic process in the    pathogenesis of age-related macular degeneration. Am J Opthalmol    2000; 130; 658-663.-   6. Vingerling J R, Dielemans I, Bots M I et al. Age-related macular    degeneration is associated with atherosclerosis. The Rotterdam    study. Am J Epidemiol 1995; 142: 404-409.-   7. Green W R and Key N N. Senile macular degeneration: a    histopathologic study. Trans Am Opthalmol soc 1977; 75; 180-254.-   8. Ambati J, Ambati B K, Yoe S H et al. Age-related macular    degeneration:etiology, pathogenesis, and therapeutic strategies.    Surv Opthalmo 2003; 48:2572-93-   9. Shima D T, Adamis A P, Ferrara N et al. Hypoxic induction of    endothelial cell growth factors in retinal cells: identification and    characterization of vascular endothelial growth factor (VEGF) as the    mitogen. Mol Med 1995; 1: 182-193.-   10. Roth F, Bindewald A, Holz F G. Keypathophysiologic pathways in    age-related macular disease. Graefes Arch Clin Exp Opthalmo 2004;    242:710-716-   11. Campochiaro P A. Retinal and choroidal neovascularisation. J    cell Physiol 2000; 97: 10242-10247,-   12. Tolentino M J, Brucker A J, Fosnot J et al. Intravitreal    injection of vascular endothelial growth factor small interfering    RNA inhibits growth and leakage in a non human primate laser-induced    model of choroidal neovascularisation. Retina 2004; 24:660-   13. Shen J, Samul R, Silva R L et al. Suppression of ocular    neovascularization with SiRNA targeting VEGF receptor. Gene ther    2005; 13:225-234-   14. Wheelan J. First clinical data on RNAi. Drug discov Today 2005;    10:1014-15-   15. Eye study group. Preclinical and phase 1A clinical evaluation of    an anti-VEGF pegylated aptamer (EYE001) for the treatment of    exudative age-related macular degeneration. Retina 2002; 22:    143-152,-   16, Krzystolik M G, Afshari M A, Adamis A P et al, Prevention of    experimental choroidal neovascularization with intravitreal    anti-vascular endothelial growth factor antibody fragments, Arch    Opthalmol 2002; 120: 338-346,-   17. Hood J D, Cheresh D A. Building a better Trap. Proc Natl Acad.    Sci USA 2003; 100: 8624-8625,-   18. Takeda A, Hata Y, Shiose S et al. Suppression of experimental    choroidal neovascularization utilizing KDR selective receptor    tyrosine kinase inhibitor Graefes Arch. Clin. exp. Opthalmol 2003;    241:1122-1129,-   19. Saishin Y, Silva R L, Callahan K et al. Periocular injection of    microspheres containing PKC412 inhibits choroidal neovascularisation    in a porcine model. Invest. Opthalmol. Vis. Sci. 2003; 44:4989-4993.-   20. Ishibashi T, Miki K, Sorgente N et al, Effects of intravitreal    administration of steroids on experimental subretinal    neovascularization in the subhuman primate. Arch Opthalmol 1985;    103; 708-711,-   21. Augustin A J, Schmidt-Erfurth U. Verteporfin therapy combined    with intravitreal triamcinolone in all types of choroidal    neovascularisation due to age-related macular degeneration.    Opthalmology 2006; 113:14-22-   22. Jonas J B, Degenring R F, Kreissig I et al. Intraocular pressure    elevation after intravitreal triamcinolone acetonide injection.    Opthalmology 2005; 112; 593-598.-   23. Slakter J S. Anecortave acetate as monotherapy for treatment of    subfoveal neovascularisation in age-related macular degeneration:    twelve-month clinical outcomes. Opthalmology 2003; 110:2372-2383.-   24. Motohiro K, Kazuhito Y, Noriaki K et al. Scavenger receptors for    oxidized lipoprotein in age-related macular degeneration Invest.    Opthalmol. Vis. Sol, 2007; 48: 1801-1807.-   25. Amburad N, Harmon C, Ibrahimi A. Membrane transport of long    chain fatty acids: evidence for a facilitated process. J. lipid Res    1998; 39: 2309-2318-   26, Endemann G, Stanton L W, Madden S et al., CD36 is a receptor for    oxidized low density lipoprotein. J. Biol. Chem 1993; 268:    11811-11816.-   27. Finnemann S C, Bonilha V L, Marmostein A D et al. Phagocytosis    of rod outer segments by retinal pigment pigment epithelium. Proc    Natl Acad Sci USA 1997; 94.12932-12937.-   28. Jimenez B, Volpert O V, Crawford S E et al, Signals leading to    apoptosis-dependent inhibition of neovascularization by    thrombospondin1. Nat med 2000; 6; 41-48.-   29. Murphy J E, Tedbury P R, Homer-Vannasinkam S et al. Biochemistry    and cell biology of mammalian scavenger receptors. Atherosclerosis    2005; 182:1-15,-   30. Pietsch A, weber C, Goretzski M et al. N-3 but not N-6 fatty    acids reduce the expression of the combined adhesion and scavenger    receptor CD36 in human monocytic cells. Cell Biochem Funct. 1995;    13: 211-216.-   31. Dithmar S, Curcio C A, Le N A at al. Ultrastructural changes in    Bruch's membrane of apolipoprotein E-deficient mice, Investigative    Opthalmology &visual Science 2000; 41; 2035-2042.-   32, Rudolf M, Ivandic B, Winkler J et al. Accumulation of lipid    particles in Bruch's membrane of LDL receptor knockout mice as a    model of age-related macular degeneration. Opthalmoloe 2004;    101:715-719,-   33. Bowers C Y. Growth hormone-releasing peptides. Cell Mol Life    Sci. 1998; 93: 1316-1329.-   34, Howard A D, Feighner S D, Cully D F et al. A receptor in    pituitary and hypothalamus that functions in growth hormone release.    Science 1996273: 974-977.-   35. De Gennaro-Colonna V, Rossoni G, Bernareggi M. et al. Cardiac    ischemia and impairment of vascular endothelium function in hearts    from growth hormone-deficient rats: protection by hexarelin. Eur J    Pharmacol. 1997; 334: 201-207.-   36. Marleau S, Harb D, Bujold K et al. EP 80317, a ligand of the    CD36 scavenger receptor, protects apolipoproteinE-deficient mice    from developing atherosclerotic lesions. FASEB J 2005; 19:1869-1871,-   37. Avallone R, Demers A, Rodrigue-Way A et al. A growth    hormone-releasing peptide that binds scavenger receptor CD36 and    ghrelin receptor upregulates ABC sterol transporters and cholesterol    efflux in macrophages through PPARγ-dependent pathway. Mol.    Endocrinol 2006;-   38, Demers A, Mc Nicoll N, Febbraio M et al. Identification of the    growth hormone-releasing peptide binding site in CD36: a    photoaffinity cross linking study. BiochemJ 2004; 382: 417-24.-   39. Boeglin, D.; Lubell, W. D. Aza-Amino Acid Scanning of Secondary    Structure Suited for Solid-Phase Peptide Synthesis with Fmoc    Chemistry and Aza-Amino Acids with Heteroatomic Side chain. J. Comb,    Chem 2005; 7(6); 864-868.-   40. Melendez, R. E.; Lubell, W. D. Aza-Amino Acid Scan for Rapid    Identification of Secondary Structure Based on N-Boc-Aza1-Dipeptides    in Peptide Synthesis. J. Am, Chem. Soc. 2004; 126; 6759-6764,-   41. Boeglin, D.; Xiang, Z.; Sorenson, N. B et al, Aza-scan of the    Potent Melanocortin Receptor Agonist. Chem. Biol. Drug Des. 2006;    67: 275-283.-   42. Rink, H. Solid-phase synthesis of protected peptide fragments    using a trialkoxy-diphenyl-methylester resin. Tetrahedron Lett,    1987, 28, 3787-390.-   43. Chang, C. D.; Meienhofer, J. Solid-phase peptide synthesis using    mild base cleavage of N alpha-fluorenylmethyloxycarbonylamino acids,    exemplified by a synthesis of dihydrosomatostatin. Int. J. Pept.    Protein Res. 1978, 11, 246.-   44. Gilbson, C.; Goodman S. L.; Hahn, D.; Hölzemann, G.; Kessler, H.    Novel Solid-Phase Synthesis of Azapeptides and Azapeptoides via    Fmoc-Strategy and Its Application in the Synthesis of    RGD-Mimetics. J. Org. Chem., 1999, 64, 7388-7394.-   45. Bodart V, Febbraio M, Demers A. et al. CD36 mediates the    cardiovascular action of growth hormone-releasing peptides in the    heart. Circ. Res 2002; 90:844-849.-   46. Mousseaux D, Le Gallic, Ryan J et al. Regulation of ERK1/2    activity by ghrelin activated growth hormone secretagogue receptor    R1A involves a PLC/PKC epsilon pathway. Brit J Pharmacol. 2006; 148;    350-365.-   47. Elizabeth Rakoczy, P., Yu, M. J., Nusinowitz, S., Chang, B., and    Heckenlively, J. R. 2006. Mouse models of age-related macular    degeneration. Exp Eye Res 82:741-752.-   48. Ershov A and Bazan N G. Induction of cyclooxygenase-2 gene    expression in retinal pigment epithelium cells by photoreceptor rod    outer segment phagocytosis and growth factors. J Neurosci Res; 1999    58:254-261.-   49. Gante, J. Azapeptides. Synthesis 1989, 405.-   50. (a) Quibell, M.; Turnell, W. G.; Johnson, T. Synthesis of    azapeptides by the Fmoc/tert-butyl/polyamide technique. J. Chem.    Soc., Perkin Trans, 1 1993, 2843-2849, (b) Liley, M.; Johnson, T.    Solid-phase synthesis of azapeptides utilizing reversible amide bond    protection to prevent hydantoin formation. Tetrahedron Lett. 2000,    41, 3983-3985.-   51. Gray, C. J.; Quibell, M.; Bagget, N.; Hammerle, T. Incorporation    of azaglutamine residues into peptides synthesized by the ultra-high    load solid (gel)-phase technique. Int. J. Pept. Protein Res, 1992,    40, 351-362.-   52. (a) Duthaler, R. O. Recent developments in the stereoselective    synthesis of α-amino acids. Tetrahedron 1994, 50, 1539-1650. (b)    Williams, R. M. in Advances in Asymmetric Synthesis; Hassner, A.,    Ed.; Jai Press Inc.: Greenwich, Conn., 1995; Vol, 1, pp 45-94. (c)    Ojima, I. in Advances in Asymmetric Synthesis; Hassner, A., Ed.; Jai    Press Inc.: Greenwich, Conn., 1995; Vol. 1, pp 95-146.-   53. O'Donnell, M. J.; Zhou, Changyou, Z., Scott, W. L. Unnatural    Peptide Synthesis. J. Am. Chem. Soc. 1996, 118, 6070-6071.-   54. Bowers, C. Y.; Momany, F. A.; Reynolds, G. A.; Hong, A.;    Newlander, K. On the in vitro and in vivo activity of a new    synthetic hexapeptide that acts on the pituitary to specifically    release growth hormone. Endocrinology 1984, 114, 1537-1545,-   55. For a recent review on growth homone secretagogues, see: (a)    Korbonits, M.; Goldstone, A. P.; Guerguiev, M.; Grossman, A. B.    Ghrelin—a hormone with multiple functions. Front Neuroendocrinol.    2004, 25, 27-68. (b) Fehrentz, J. A.; Martinez, J.; Boeglin, D.;    Guerlavais, V.; Deghenghi, R. I. Growth hormone secretagogues: past,    present and future. Drugs 2002, 5, 804-814.-   56. Adams, J. H.; et al. A Reinvestigation of the Preparation,    Properties, and Applications of Aminomethyl and    4-Methylbenzhydrylamine Polystyrene Resins. J. Org. Chem. 1998, 63,    3706-3716.-   57. Torrini, I.; Zecchini, G. P.; Paradisi, M. P.; Mastropietro, G.;    Lucente, G.; Gavuzzo, E.; Mazza, F. Topographically constrained    aromatic α-aza-amino acids. Part 2. New azaTic-containing peptides:    synthesis, conformation, and intramolecular NH—N interaction.    Tetrahedron 1999, 55, 2077-2090.-   58. Wieczerzak, E.; Drabik, P.; Lankiewicz, L.; Oldziej, S.;    Grzonka, Z.; Abrahamson, M.; Grubb, A.; Bromme, D. Azapeptides    Structurally Based upon Inhibitory Sites of Cystatins as Potent and    Selective Inhibitors of Cysteine Proteases. J. Med, Chem. 2002, 45,    4202-4211.-   59. (a) Arrowsmith, J. E.; Cook, M. J.; Hardstone, D. J. Reactions    of anions of N-benzylidenebenzylamines and related compounds. A    simple route to α-substituted benzylamines. J. Chem. Soc., Perkin    Trans, 1 1979, 2364-2368. and references cited therein, (b) Asai,    T.; Aoyama, T.; Shioiri, T. New methods and reagents in organic    synthesis. 7. α-Alkylation of benzylamine under phase-transfer    catalyzed conditions. Synthesis 1980, 811-812., (c) Bradamante, S.;    Ferraccioli, R.; Pagani, G. A. The reaction of sodium    1,3-diphenyl-2-azapropenide with 1,2-epoxycyclohexane. J. Chem.    Soc., Perkin Tram. 1, 3, 1987, 515-518.-   60. O'Donnell, M. J.; et. al. Acidities of glycine Schiff bases and    alkylation of their conjugate bases J. Am. Chem. Soc. 1998, 110,    8520-8525,-   61. O'Donnell, M. J.; Delgado, F.; Hostettler, C.; Schwesinger, R.    An efficient homogeneous catalytic enantioselective synthesis of    α-amino acid derivatives. Tetrahedron Lett. 1998, 39, 8775-8778.-   62. (a) Sturino, C. F.; Fallis, A. G. Samarium(II) Iodide Induced    Radical Cyclizations of Halo- and Carbonylhydrazones. J. Am. Chem.    Soc. 1994, 116, 7447-7448. (b) Mino, T.; Yamashita, M. Synthesis of    2-alkyl-2-methyl-3-butenonitriles via alkylation of    2-methyl-2-butenal N,N-dimethylhydrazone. J. Org. Chem. 1996, 61,    1159-1160. (c) Nakamura, E.; Kubota, K.; Sakata, G. Addition of    Zincated Hydrazone to Vinyl Grignard Reagent. Ketone Synthesis by    One-Pot Assembly of Four Components. J. Am. Chem. Soc. 1997, 119,    5457-5458.-   63. Enders, D.; Wortmann, L.; Peters, R. Recovery of Carbonyl    Compounds from N,N-Dialkylhydrazones. Acc. Chem. Res. 2000, 33,    157-169,-   64. Hart, T. W.; Guillochon, D.; Perrier, G.; Sharp, B. W.; Toft, M.    P.; Vacher, B.; Walsh, R. J. A. The synthesis of RP 66471. A potent    potassium channel opener. Tetrahedron Lett, 1992, 33, 7211-7214.-   65. Diez, E.; Lopez, A. M.; Pareja, C.; Martin, E.; Fernandez, R.;    Lassaletta, J. M. Direct synthesis of dithioketals from    N,N-dialkylhydrazones. Tetrahedron Lett. 1998, 39, 7955-7958.-   66. Wilson, R. D.; Watson, S. P; Richards, S. A. Solid phase    synthesis of 5-aminopyrazoles and derivatives. Part II. Tetrahedron    Lett, 1998, 39, 2827-2830.-   67. Falb, E.; Yechezkel, T.; Salitra, Y.; Gilon, C. In situ    generation of Fmoc-amino acid chlorides using    bis-(trichloromethyl)carbonate and its utilization for difficult    couplings in solid-phase peptide synthesis. J. Pept. Res. 1999, 53,    507-517.-   68. (a) Meienhofer, J.; Waki, M.; Heimer, E. D.; Lambros, T. J.;    Makofske, R. C.; Chang, C. D. Solid phase synthesis without    repetitive acidolysis. Preparation of leucyl-alanyl-glycyl-valine    using 9-fluorenylmethyloxycarbonylamino acids. Int, J. Pept. Protein    Res. 1979, 13, 35-42. (b) Lubell, W. D.; Blankenship, J. W.;    Fridkin, G.; Kaul, R. Product class 11: Peptides. Science of    Synthesis 2005, 21, 713-809,-   69. Yang, J.; Song, H.; Xiao, X.; Wang, J.; Qin; Y. Biomimetic    approach to perophoramidine and communesin via an intramolecular    cyclopropanation reaction. Org. Lett. 2006, 8(10), 2187-2190.-   70. Kaiser, E.; Colescott, R. L.; Bassinger, C. D.; Cook, P. I.    Color test for detection of free terminal amino groups in the    solid-phase synthesis of peptides, Anal. Biochem, 1970, 34, 595-598.

All literature, patents, published patent applications cited herein arehereby incorporated by reference,

One or more currently preferred embodiments have been described by wayof example. It will be apparent to persons skilled in the art that anumber of variations and modifications can be made without departingfrom the scope of the invention as defined in the claims.

The invention claimed is:
 1. A method for reducing CD36-relatedinflammation and/or angiogenesis in a subject in need thereof, saidmethod comprising administering to said subject an effective amount ofan azapeptide analogue of GHRP-6 of Formula I.3, wherein said azapeptideanalogue binds to CD36 and exhibits reduced binding to the growthhormone secretagogue receptor-1a relative to GHRP-6:A-Xaa₁-Xaa₂-Xaa₃-N(R^(A))—N(R^(B))—C(O)-Xaa′₃-Xaa′₄-B  I.3 wherein Xaa₁is His, D-His, Ala, D-Ala, Phe or D-Phe; Xaa₂ is Trp or D-Trp; Xaa₃ isAla or D-Ala; Xaa′₃ is Phe, D-Phe, Ala or D-Ala; Xaa′₄ is Lys, D-Lys,Ala or D-Ala; and not more than one of Xaa₁, Xaa′₃ and Xaa′₄ is Ala orD-Ala; A is 1) H, 2) C₁-C₆ alkyl, 3) C₂-C₆ alkenyl, 4) C₂-C₄ alkynyl, 5)C₃-C₇ cycloalkyl, 6) haloalkyl, 7) heteroalkyl, 8) aryl, 9) heteroaryl,10) heteroalkyl, 11) heterocyclyl, 12) heterobicyclyl, 13) C(O)R³, 14)SO₂R³, 15) C(O)OR³, or 16) C(O)NR⁴R⁵, wherein the alkyl, the alkenyl,the alkynyl and the cycloalkyl are optionally substituted with one ormore R¹ substituents; and wherein the aryl, the heteroaryl, theheterocyclyl and the heterobicyclyl are optionally substituted with oneor more R² substituents; B is 1) OH, 2) OR³, or 3) NR⁴R⁵; R^(A) andR^(B) are independently chosen from 1) H, 2) C₁-C₆ alkyl, 3) C₂-C₆alkenyl, 4) C₂-C₆ alkynyl, 5) C₃-C₇ cycloalkyl, 6) C₅-C₇ cycloalkenyl,7) haloalkyl, 8) heteroalkyl, 9) aryl, 10) heteroaryl, 11)heterobicyclyl, or 12) heterocyclyl, wherein the alkyl, alkenyl, alkynyland the cycloalkyl and cycloalkenyl are optionally substituted with oneor more R¹ substituents; and wherein the aryl, the heteroaryl, theheterocyclyl and the heterobicyclyl are optionally substituted with oneor more R² substituents, or alternatively, R^(A) and R^(B) together withthe nitrogen to which each is bonded form a heterocyclic or aheterobicyclic ring; R¹ is 1) halogen, 2) NO₂, 3) CN, 4) haloalkyl, 5)C₃-C₇ cycloalkyl, 6) aryl, 7) heteroaryl, 8) heterocyclyl, 9)heterobicyclyl, 10) OR⁶, 11) S(O)₂R³, 12) NR⁴R⁵, 13) NR⁴S(O)₂R³, 14)COR⁶, 15) C(O)OR⁶, 16) CONR⁴R⁵, 17) S(O)₂NR⁴R⁵, 18) OC(O)R⁶, 19)SC(O)R³, 20) NR⁶C(O)NR⁴R⁵, 21) heteroalkyl, 22) NR⁶C(NR⁶)NR⁴R⁵, or 23)C(NR⁶)NR⁴R⁵; wherein the aryl, heteroaryl, heterocyclyl, andheterobicyclyl are optionally substituted with one or more R²substituents; R² is 1) halogen, 2) NO₂, 3) CN, 4) C₁-C₆ alkyl, 5) C₂-C₆alkenyl, 6) C₂-C₄ alkynyl, 7) C₃-C₇ cycloalkyl, 8) haloalkyl, 9) OR⁶,10) NR⁴R⁵, 11) SR⁶, 12) COR⁶, 13) C(O)OR⁶, 14) S(O)₂R³, 15) CONR⁴R⁵, 16)S(O)₂NR⁴R⁵, 17) aryl, 18) heteroaryl, 19) heterocyclyl, 20)heterobicyclyl, 21) heteroalkyl, 22) NR⁶C(NR⁶)NR⁴R⁵, or 23) C(NR⁶)NR⁴R⁵,wherein the aryl, the heteroaryl, the heterocyclyl, and theheterobicyclyl are optionally substituted with one or more R⁷substituents; R³ is 1) C₁-C₆ alkyl, 2) C₂-C₆ alkenyl, 3) C₂-C₄ alkynyl,4) C₃-C₇ cycloalkyl, 5) haloalkyl, 6) aryl, 7) heteroaryl, 8)heterocyclyl, or 9) heterobicyclyl, wherein the alkyl, the alkenyl, thealkynyl and the cycloalkyl are optionally substituted with one or moreR¹ substituents; and wherein the aryl, the heteroaryl, the heterocyclyland the heterobicyclyl are optionally substituted with one or more R²substituents; R⁴ and R⁵ are independently chosen from 1) H, 2) C₁-C₆alkyl, 3) C₂-C₆ alkenyl, 4) C₂-C₆ alkynyl, 5) aryl, 6) heteroaryl, or 7)heterocyclyl, or R⁴ and R⁵ together with the nitrogen to which they arebonded form a heterocyclic ring; R⁶ is 1) H, 2) C₁-C₆ alkyl, 3) C₂-C₆alkenyl, 4) C₂-C₆ alkynyl, 5) aryl, 6) heteroaryl, or 7) heterocyclyl;R⁷ is 1) halogen, 2) NO₂, 3) CN, 4) C₁-C₆ alkyl, 5) C₂-C₆ alkenyl, 6)C₂-C₄ alkynyl, 7) C₃-C₇ cycloalkyl, 8) haloalkyl, 9) OR⁶, 10) NR⁴R⁵, 11)SR⁶, 12) COR⁶, 13) C(O)OR⁶, 14) S(O)₂R³, 15) CONR⁴R⁵, 16) S(O)₂NR⁴R⁵,17) heteroalkyl, 18) NR⁶C(NR⁶)NR⁴R⁵, or 19) C(NR⁶)NR⁴R⁵; or apharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein Xaa₁ is His, D-His, Ala or D-Ala.
 3. The method of claim 1,wherein Xaa₁ is His or D-His.
 4. The method of claim 1, wherein Xaa₂ isD-Trp.
 5. The method of claim 1, wherein Xaa₃ is Ala.
 6. The method ofclaim 1, wherein Xaa′₃ is D-Phe.
 7. The method of claim 1, wherein Xaa′₄is Lys.
 8. The method of claim 1, wherein Xaa₁ is Ala or His, Xaa₂ isDTrp, Xaa₃ is Ala, Xaa′₃ is DPhe, and Xaa′₄ is Lys.
 9. The method ofclaim 8, wherein said azapeptide analogue is:A-His-DTrp-Ala-AzaPhe-DPhe-Lys-B; A-Ala-DTrp-Ala-AzaTyr-DPhe-Lys-B;A-Ala-DTrp-Ala-AzaPhe-DPhe-Lys-B;A-His-DTrp-Ala-Aza(p-bromo)Phe-DPhe-Lys-B;A-His-D-Trp-Ala-AzaTyr-DPhe-Lys-B;A-His-DTrp-Ala-Aza(p-fluor)Phe-DPhe-Lys-B;A-His-DTrp-Ala-Aza(p-OCH₃)Phe-DPhe-Lys-B; orA-His-DTrp-Ala-AzaLys-DPhe-Lys-B.
 10. The method of claim 9, wherein Ais H and B is NH₂.
 11. The method of claim 9, wherein the azapeptideanalogue is Ala-DTrp-Ala-AzaTyr-DPhe-Lys-NH₂.
 12. The method of claim 9,wherein the azapeptide analogue is Ala-DTrp-Ala-AzaPhe-DPhe-Lys-NH₂. 13.The method of claim 9, wherein the azapeptide analogue isHis-DTrp-Ala-AzaPhe-DPhe-Lys-NH₂.
 14. The method of claim 9, wherein theazapeptide analogue is His-DTrp-Ala-Aza(p-brorno)Phe-DPhe-Lys-NH₂. 15.The method of claim 9, wherein the azapeptide analogue isHis-D-Trp-Ala-AzaTyr-DPhe-Lys-NH₂.
 16. The method of claim 9, whereinthe azapeptide analogue is His-DTrp-Ala-Aza(p-fluor)Phe-DPhe-Lys-NH₂.17. The method of claim 9, wherein the azapeptide analogue isHis-DTrp-Ala-Aza(p-OCH₃)Phe-DPhe-Lys-NH₂.
 18. The method of claim 9,wherein the azapeptide analogue is His-DTrp-Ala-AzaLys-DPhe-Lys-NH₂. 19.The method of claim 1, wherein said subject suffers from atherosclerosisor an atherosclerotic cardiovascular disease.
 20. The method of claim 1,wherein said subject suffers from age-related macular degeneration.